Delivery of nucleic acid to cells

ABSTRACT

Nucleic acids are compacted, substantially without aggregation, to facilitate their uptake by target cells of an organism to which the compacted material is administered. The nucleic acids may achieve a clinical effect as a result of gene expression, hybridization to endogenous nucleic acids whose expression is undesired, or site-specific integration so that a target gene is replaced, modified or deleted. The targeting may be enhanced by means of a target cell-binding moiety. The nucleic acid is preferably compacted to a condensed state.

This invention was made with government support under DK21859, DK25541,DK43999 and HL53672 awarded by the National Institute of Health. Thegovernment has certain rights in the invention.

This application is a Continuation of PCT Application No. PCT/US95/03677filed on Mar. 23, 1995 which is a Continuation-In-Part of applicationSer. No. 08/216,534, filed Mar. 23, 1994, now abandonded, herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the in vivo delivery of exogenousnucleic acids to cells of multicellular organisms.

BACKGROUND

Functional exogenous genes can be introduced to mammalian cells in vitroby a variety of physical methods, including transfection, directmicroinjection, electroporation, and coprecipitation with calciumphosphate. Most of these techniques, however, are impractical fordelivering genes to cells within intact animals.

Receptor-Mediated Uncompacted DNA Delivery In Vivo

Receptor-mediated gene transfer has been shown to be successful inintroducing transgenes into suitable recipient cells, both in vitro andin vivo. This procedure involves linking the DNA to a polycationicprotein (usually poly-L-lysine) containing a covalently attached ligand,which is selected to target a specific receptor on the surface of thetissue of interest. The gene is taken up by the tissue, transported tothe nucleus of the cell and expressed for varying times. The overalllevel of expression of the transgene in the target tissue is dependenton several factors: the stability of the DNA-carrier complex, thepresence and number of specific receptors on the surface of the targetedcell, the receptor-carrier ligand interaction, endocytosis and transportof the complex to the nucleus, and the efficiency of gene transcriptionin the nuclei of the target cells.

Wu, et al., U.S. Pat. 5,166,320, discloses tissue-specific delivery ofDNA using a conjugate of a polynucleic acid binding agent (such aspolylysine, polyarginine, polyornithine, histone, avidin, or protamine)and a tissue receptor-specific protein ligand. For targeting livercells, Wu suggests "asialoglycoprotein (galactose-terminal) ligands".

Wagner, et al., Proc. Natl. Acad. Sci., 88:4255-4259 (1991) and U.S.Pat. No. 5,354,844 disclose complexing a transferrin-polylysineconjugate with DNA for delivering DNA to cells via receptor mediatedendocytosis. Wagner, et al., teach that it is important that there besufficient polycation in the mixture to ensure compaction of plasmid DNAinto toroidal structures of 80-100 nm diameter, which, they speculate,facilitate the endocytic event.

Direct Injection of Naked, Uncompacted DNA

The possibility of detecting gene expression by directly injecting nakedDNA into animal tissues was demonstrated first by Dubenski et al., Proc.Nat. Acad. Sci. USA, 81:7529-33 (1984), who showed that viral or plasmidDNA injected into the liver or spleen of mice was expressed atdetectable levels. The DNA was precipitated using calcium phosphate andinjected together with hyaluronidase and collagenase. The transfectedgene was shown to replicate in the liver of the host animal. Benvenistyand Reshef, Proc. Nat. Acad. Sci. USA, 83:9551-55 (1986) injectedcalcium phosphate precipitated DNA intraperitoneally into newborn ratsand noted gene expression in the livers of the animals 48 hours aftertransfection. In 1990, Wolff et al., Science, 247:1456-68 (1990),reported that the direct injection of DNA or RNA expression vectors intothe muscle of mice resulted in the detectable expression of the genesfor periods for up to 2 months. This technique has been extended byAcsadi et al., New Biologist, 3:71-81 (1991) to include direct injectionof naked DNA into rat hearts; the injected genes were expressed in theheart of the animals for up to 25 days. Other genes, including the genefor dystrophin have been injected into the muscle of mice using thistechnique. This procedure forms the base of a broad approach for thegeneration of immune response in an animal by the administration of agene by direct injection into the target tissue. The gene is transientlyexpressed, producing a specific antigen. (See Donnelly et al., TheImmunologist, 21, pp. 20-26 (1994) for a recent review). However, theDNA used in these experiments has not been modified or compacted toimprove its survival in the cell, its uptake into the nucleus or itsrate of transcription in the nucleus of the target cells.

SUMMARY OF THE INVENTION

The present invention relates to the in vivo delivery of exogenousnucleic acids to cells of multicellular organisms. When the nucleic acidincludes an expressible gene, that gene can be expressed in the cell. Insome embodiments, a tissue-specific carrier molecule is prepared, whichis a bifunctional molecule having a nucleic acid-binding moiety and atarget tissue-binding moiety.

The nucleic acid can be compacted at high concentrations with thecarrier molecule at a critical salt concentration. The nucleicacid-loaded carrier molecule is then administered to the organism.

In one embodiment, the present invention contemplates a method fordelivering an oligonucleotide to cells of an animal, comprising: a)providing: i) a target binding moiety capable of binding to a polymericimmunoglobulin receptor present on the surface of a cell in a tissue ofan animal; ii) a nucleic acid binding moiety; iii) an expression vectorcomprising an oligonucleotide encoding one or more gene products; iv) arecipient animal; b) conjugating said target binding moiety to saidnucleic acid binding moiety to form a carrier; c) coupling said carrierwith said expression vector to form a pharmaceutical composition; and d)administering said composition to said recipient animal. It ispreferrred that said expression vector (i.e., nucleic acid) iscompacted.

In a preferred embodiment, the oligonuceotide is delivered to a tissueselected from the group consisting of lung, trachea and liver tissue. Inparticularly preferred embodiment, the oligonuceotide is delivered to amucosal epithelial cell.

In a preferred embodiment, the expression vector further comprises apromoter sequence operably linked to the oligonucleotide encoding one ormore gene products. The invention is not limited by the nature of thepromoter sequence chosen; any promoter sequence which is functional incells expressing the polymeric immunoglobulin receptor may be utilized.In a particularly preferred embodiment, the promoter sequence is a viralpromoter sequence. The viral promoter is preferably selected from thegroup consisting of the SV40 promoter, the MMTV promoter and the CMVpromoter.

In one embodiment, said target binding moiety is an antibody directedagainst the secretory component of a mammalian polymeric immunoglobulinreceptor. It is preferred that said antibody is a monoclonal antibody.

It is not intended that the present invention be limited by the natureof the nucleic acid binding moiety. In one embodiment, the nucleic acidbinding moiety is a polycation, such as poly-L-lysine.

It is also not intended that the present invention be limited by thenature of the administration of the composition. In one embodiment, saidadministering comprises injection of said composition into saidrecipient animal (e.g., by intravenous injection).

The present invention can be used with success with a variety ofanimals. Particular therapeutic success is achieved with humans. In thatregard, it may be desireable, following injection of said composition,to examine the relevant tissue containing said polymeric immunoglobulinreceptor on their surface for the expression of one or more of said geneproducts encoded by said expression vector.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-J--Physical characterization of the galactose-poly-L-lysine/DNAcomplexes. FIG. 1A shows CD spectra associated with normal DNA insolution and with certain poly-L-lysine/DNA complexes. Sixty microgramsof RNA-free CMV-β-galactosidase plasmid (dissolved in TE buffer, pH 8),150 μl of 700 mM NaCl were vortexed at medium speed in a VIBRAXapparatus (IKA-VIBRAX-VXR). Nineteen micrograms ofα-galactopyranosyl-phenyl isothiocyanate/poly-L-lysine biconjugate in150 μl of 700 mM NaCl were added dropwise to the vortexing solution ofDNA. The slow addition of the polycation results in the formation of aturbid solution which is dissolved by the slow, stepwise addition of 3μl aliquots of 5 M NaCl. The disappearance of the turbidity wasmonitored by eye and the solutions of DNA/poly-L-lysine complexes wereinvestigated by CD. At this point (0.97 M NaCl), the CD spectrum wasthat characteristic of aggregated DNA. Further addition of 2 μl aliquotsof 5 M NaCl (resulting in a concentration of 1.031 M NaCl) yielded theCD spectrum expected for a condensed (or a relaxed) DNA complex. The CDspectrum of uncomplexed double stranded DNA at 1M NaCl was also taken.The spectra were obtained using a JASCO-600 spectropolarimeter with a0.1 cm cuvette. The spectrum of the buffer was subtracted in each case.

FIGS. 1B-1G are electronic micrographs (EM). 1B-1D, 1F and 1G are takenat 300,000×. The bar in 1D represents 33.3 nm. FIG. 1E was taken at600,000×, and the bar is 16.6 nm long. Uranyl acetate staining wasperformed as previously described. (Ennever, et al., Biochem. Biophys.Acta, 826:67 (1985)). Briefly, the grid was subjected to glow dischargeprior to staining. A drop of DNA solution was added to the grid, blottedand stained using 0.04% uranyl acetate.

For the EM studies shown in FIGS. 1B-1F, 60 μg of PEPCK-hFIX plasmid DNA(dissolved in TE buffer, pH 8), in 150 μl of 700 mM NaCl were vortexedat medium speed in a VIBRAX apparatus (IKA-VIBRAX-VXR). Nineteenmicrograms of α-galactopyranosyl-phenyl isothiocyanate/poly-L-lysinebioconjugate in 150 μl of 700 mM NaCl were added dropwise to thevortexing solution of DNA. The slow addition of the polycation resultsin the formation of a turbid solution which is dissolved by the slow,stepwise addition of 3 μl aliquots of 5 M NaCl. The disappearance of theturbidity was monitored by eye and the solution of DNA/poly-L-lysinecomplexes was investigated by EM (FIG. 1C). Further addition of 2 μlaliquots of 5 M NaCl resulted in structural changes as shown in FIGS. 1Dand 1E.

FIG. 1B is an EM of uncomplexed DNA (1 μg/ml at 1M NaCl). FIG. 1Cdepicts a DNA complex at a suboptimal concentration of NaCl (760 mM).The DNA is in the aggregated state; clusters of unimolecular toroids arevisible. In FIG. 1D the DNA complex is at an optimal concentration ofNaCl for the complex in question (968 mM). The DNA is properlycondensed; only individual toroids can be seen. For FIG. 1E, fourcomplexes of DNA from FIG. 1D were selected and printed at highermagnification.

In FIG. 1F, we see a DNA complex, at a concentration of 1.068 M NaCl,which is above optimal for condensation of this complex. The DNA is inthe relaxed state. Note the branched unimolecular toroids in which anucleus of condensation is visible and the rod-like DNA fibers.

Differences in concentration of NaCl required for aggregated, condensed,and relaxed states in the above experiments represent DNA or polycationspecific differences.

In a third experiment, complexes of CMV-β-galactosidase andgalactosylated poly-L-lysine were formed essentially as in Wu et al.Briefly, plasmid DNA and galactosylated poly-L-lysine were combined in 3M NaCl. The samples were incubated for 1 hour at room temperature, thendialyzed against 0.15 M NaCl for 16 hr through membranes with a3,500-dalton molecular mass limit. On visual inspection, no precipitateswere present in the dialysate.

FIG. 1G is an electron micrograph of the resulting DNA complex, which isin the multimolecular aggregated state. Note that the toroids here arelarger than in 1C or 1D (the scale is the same). FIG. 1H shows the CDspectrum from 240 to 300 nm for uncomplexed DNA and for aggregatedmultimolecular DNA/poly-L-Lys complexes, so as to highlight theinversion of the normal DNA spectrum maximum at 269 nm. This inversionis characteristic of multimolecular aggregation.

In another experiment, sixty micrograms of PEPCK-hFIX plasmid DNA(dissolved in TE buffer, pH 8), in 150 μl of 200 mM NaCl were vortexedat medium speed in a VIBRAX apparatus (IKA-VIBRAX-VXR). Nineteenmicrograms of α-galactopyranosyl-phenyl isothiocyanate/poly-L-lysinebiconjugate in 150 μl of 200 mM NaCl were added dropwise to thevortexing solution of DNA. The addition of the polycation resulted inthe formation of precipitates on visual inspection.

FIG. 1I is a CD spectrum, given by a precipitated DNA complex. It isessentially flat from 240 to 300 nm. FIG. 1J is an electron micrographof the precipitated DNA.

FIGS. 2A & B--Functional relevance and specificity of the gene transfersystem. (A) The relative concentration of human factor IX in the bloodof animals treated with the DNA complex was evaluated by measuring theprocoagulant activity of human factor IX. A modification of the onestage, kaolin-activated, partial thromboplastin time with factorIX-deficient human plasma was used. Blood samples were obtained fromexperimental animals by venipuncture. One fiftieth volume of 500 mMsodium citrate, pH 5.0, was added to prevent coagulation, and the plasmawas stored at -20° C. The samples were assayed in duplicate, and theiractivity was compared to the functional activity of pooled plasma from24 normal adult human males. In all calculations, one unit of factor IXactivity in one ml of normal human plasma is equivalent to 100%functional activity or approximately 3 μg of factor IX per ml.Background human factor IX activity in the rat plasma was subtractedprior to graphic representation. (B) Transfected animals were fed acarbohydrate-free/high protein diet for one week. Blood samples weretaken at the initiation of the treatment and after one week on the dietand analyzed by Western blot hybridization. The animals at 8 and 12 dayswere compared with transfected rats fed a standard chow diet. The datawere obtained by densitometric analysis of Western blot photographicfilms and indicate fold increase in human factor IX protein after thedietary treatment.

FIG. 3--Tissue specificity of mannosylated DNA complex in targeting DNAto the macrophages in vivo. Mannosylated poly-L-lysine was conjugated toSV40/luciferase DNA. 300 μg of the DNA complex were introduced into thecaudal vena cava of rats. Four days after injection tissue extracts weremade and assayed for luciferase activity. The luciferase activity isplotted as Integrated Light Units per milligram of protein extract fromspleen, liver and lung. In other tissues no activity was found. Data areexpressed as means±standard error of the mean (SEM). The light bars arethe non-transfected controls (n=4), and the dark bars, animalstransfected with mannosylated poly-L-lysine/DNA complexes (n=5).

FIG. 4--Specificity of mannosylated DNA complex in targeting DNA toprimary culture of macrophages in vitro. Primary cultures of peritonealmacrophages were transfected with either galactosylated poly-L-lysine(light bars) or mannosylated poly-L-lysine (dark bars) conjugated to aSV40/luciferase DNA. At the indicated times (2, 4, 8, and 24 hours)cells were washed. Twenty-four hours after transfection, cells wereharvested and assayed for luciferase activity. The luciferase activityis plotted as Relative Luciferase Activity after being standardized bythe activity found in untransfected controls. Data are expressed asmeans±standard error of the mean (SEM).

FIG. 5--Competition between the mannosylated DNA complex andmannosylated bovine serum albumin for binding to the Mannose receptor ofmacrophages. Primary culture of peritoneal macrophages were transfectedwith mannosylated poly-L-lysine conjugated to SV40/luciferase DNA (T).Prior to the addition of the DNA complex a 100-fold excess mannosylatedbovine serum albumin was added to one set of plates (Tc).Non-transfected controls (NT) were also assayed for luciferase activity24 hours after transfection. The luciferase activity is plotted asRelative Luciferase Activity after being standardized relative to theactivity found in untransfected controls. Data are expressed asmeans±standard error of the mean (SEM).

FIG. 6--In vivo gene transfer using the anti-rat plg-R Fab-poly-L-lysineconjugated DNA complex. Fab-poly-L-lysine was conjugated toSV40/luciferase DNA and introduced into the caudal vena cava of rats(Transfected) (n=3). Untransfected controls (Control) (n=3), animalsinjected with an Fab-poly-L-lysine-DNA complex containing an Fabfragment obtained from an irrelevant IgF (IFab) (n=3), and animalsinjected with a DNA complex that does not contain an SV40/Luciferasegene (IDNA) (n=3), were run as controls. Two days after injection tissueextracts were prepared and assayed for luciferase activity. Theluciferase activity is plotted as Integrated Light Units per milligramof protein extract. Data are expressed as means±standard error of themean (SEM).

FIG. 7--Time-course of expression in lung and liver of animals injectedusing the anti-rat plg-R Fab-poly-L-lysine conjugated DNA complex.Fab-poly-L-lysine was conjugated to SV40/luciferase DNA and introducedinto the caudal vena cava of rats (n=9). Rats were killed 2 (n=3), 4(n=3) and 6(n=3) days after injection. Lung and liver extracts wereprepared and assayed for luciferase activity. The luciferase activity isplotted as Integrated Light Units per milligram of protein extract usinga logarithmic scale. Data are expressed as means±standard error of themean (SEM).

FIG. 8--Competition between the galactoslyated DNA complex andasialoorosomucoid for binding to the ASGP receptor of HepG2 cells. HepG2hepatoma cells were transfected with galactosylated poly-L-lysineconjugated to PEPCK-hFIX DNA. Prior to the addition of the DNA complex a100-fold excess asialoorosomucoid was added to one set of plates(+Comp.). DNA internalization was monitored by slot-blot hybridizationof the culture medium containing the DNA complex. Data are expressed aspercentage of DNA internalized by the receptor at different times aftertransfection.

FIG. 9--Direct injection to the muscle and liver of naked DNA vs.condensed DNA. One hundred micrograms of naked DNA encodingSV40-luciferase were injected into the liver and abdominal muscle of tworats. The same amount of the pSV40-luciferase plasmid complexed topoly-L-lysine and condensed as described in Example 1 was injected aswell into the liver and abdominal muscle of another two animals. Ratswere sacrificed 48 hours post-injection. A piece of liver and abdominalmuscle were homogenized in lysis buffer and cell lysates were analyzedfor luciferase activity. All luciferase measurements were performed intriplicate, expressed as an average of the values and standardized fortotal protein. FIG. 9 shows the integrated luciferase units per mg ofprotein in the two different sets of animals.

FIG. 10--Direct injection into the brain tectum of naked DNA vs.condensed DNA. Intratectal injections of naked and poly-L-lysinecondensed plasmid DNA can achieve high levels of expression in the cellbody of the neuron over 20 days. β-galactosidase activity in retinasfrom rats whose brains were injected into the tectal areas andadministered with either naked pCMV-lacZ, or condensed pCMV-lacZ(pCMV-lacZ+lys) at the concentrations shown. When the DNA is notcondensed with poly-L-lysine the level of expression returns tobackground after 10 days post-injection.

FIG. 11--Changes in the absorbance of the DNA complexes during thecondensation process. A plasmid containing the chimeric CMV-hLDLreceptor gene was condensed with poly-L-lysine, using the proceduredescribed in detail in Example 1. After the addition of poly-L-lysinethe absorbance of the solution at 260 nm was determined. ConcentratedNaCl was then added stepwise and the absorbance determined. The expectedabsorbance for the DNA contained in the complex is indicated by thedotted line. The initial NaCl concentration used in the condensationreaction was 500 mM.

FIG. 12--Relationship between the structure of the DNA complex and itsfunction in adult rats. DNA-galatosylated poly-lysine complexes wereprepared which correspond to various states of condensation/aggregationshown in FIGS. 1B-1G. The DNA consisted of the SV40 promoter linked tothe structural gene for P. pyralis luciferase gene. Rats were injectedin the caudal vena cava with 300 μg of the various DNA complexes and theactivity of luciferase was determined in extracts from the liver and thespleen 48 hr after injection. Each bar represents the mean±SEM for threerats; control rats were not injected with the DNA complex.

FIG. 13--Introduction of 3 mg of PEPCK-hLDLr in its relaxed(noncomplexed) vs. condensed form. In order to introduce the DNA complexinto the animal, we perform a single injection of 3-10 ml of theDNA-complex solution (˜400-900 mM NaCl) into the marginal ear vein ofthe rabbit. Approximately 1.5 ml of blood was drawn at the timesindicated from the ear artery at 4 p.m. The determination of theconcentration of serum cholesterol was performed in the ClinicalLaboratory of University Hospitals of Cleveland from 300 μl of serum.The administration of a DNA complex solution containing 3 mg of thepPEPCK-hLDLR plasmid in a relaxed state to rabbit #676 did not result ina significant decrease (first arrow) in total serum cholesterol levels.A second injection of DNA complexes appropriately condensed containing 3mg of the same DNA (second arrow) caused a 20% reduction of the levelsof cholesterol in the blood. Four weeks after this secondadministration, cholesterol returned to approximately pre-treatmentlevels, reaching a peak at about 35 days.

FIG. 14--Injection of the poly-L-lysine/DNA complex containing 9 mg ofthe chimeric PEPCK-hLDLr gene. In our second experiment, 9 mg of thePEPCK-hLDLr gene appropriately condensed with galactosylatedpoly-L-lysine were administered to rabbit #737. As shown in FIG. 14, thetreatment resulted in a 38% reduction of total serum cholesterol levelswhich lasted for about 5 weeks. Thus, a 3-fold increase in the dose ofDNA complex resulted in a 2-fold reduction in total serum cholesterollevels.

FIG. 15--Injection of the poly-L-lysine/DNA complex containing 3 mg ofthe chimeric CMV-hLDLr gene. The admcontaining of a DNA complex solutioncontaining 3 mg of the chimeric CMV-hLDL receptor gene to rabbit #16resulted in a maximal reduction of 30% in total serum cholesterol levels(FIG. 15). Eleven weeks after the injection, cholesterol levels arestill 20% below those observed before the treatment.

FIGS. 16A & B--Injection of multiple doses of the poly-L-lysine/DNAcomplex containing 3 mg of the chimeric CMV-hLDLr gene. Rabbits #775(FIG. 16A) and #774 (FIG. 16B) were injected with 3 mg of the pCMV-hLDLRcomplex. In rabbit #775, this caused a maximal 24% reduction incholesterol concentration in the blood, 3 weeks after treatment. Twoadditional injections did not result in a further significant reductionin serum cholesterol. In Rabbit #774, we observed a 36% decrease in thecholesterol levels in the blood (FIG. 16B) after the initial injeciton.Four reinjections once every 2 weeks were performed with the same amountof DNA complex. Two of them resulted in a minimal effect while the othertwo in a null reduction of total serum cholesterol levels. However,after five administrations of the DNA complex solution containing 3 mgof pCMV-hLDLr, the concentration of cholesterol had dropped about 48%with respect to pre-treatment levels.

Rabbit #774 was then treated with 10 mg of lovastatin (striped bar) perday for 10 weeks. A further 20% reduction in the levels of cholesterolhas been observed. The inhibition of the endogenous pathway forcholesterol synthesis has thus brought the cholesterol concentration ofrabbit #774 to 40% of that prior to the first gene transfer (FIG. 16B).

FIG. 17--Mock-injection of the poly-L-lysine/DNA complex containing 3 mgof the chimeric SV40-luciferase gene (irrelevant DNA). In order tocontrol for a possible nonspecific reduction in total serum cholesterollevels by injecting rabbits with the galactosylated poly-L-lysine/DNAcomplexes in a solution with high NaCl concentration (˜900 mM), we haveadministered a DNA complex solution containing an irrelevant DNA such asthe luciferase gene into rabbit #775. FIG. 17 shows that the injectionresults in a non-significant (≦12%) and transient (≦5 days) reduction inthe serum cholesterol concentration. Thus, we have confirmed that thereduction in total serum cholesterol levels after the injection ofappropriately condensed DNA particles encoding the human LDL receptorgene are not a result of either the high NaCl concentration of thesolution or the presence of galactosylated poly-L-lysine/DNA particles.

FIG. 18--Relationship of turbidity to NaCl concentration. The figureshows the effect of initial and current NaCl concentration on theturbidity of a DNA/poly-lysine solution. Each line represents adifferent initial concentration.

FIG. 19--Effect of poly-L-lysine length on condensation concentration ofNaCl.

FIGS. 20A-E--CD spectra for different complexes. CD spectra were takenin a 0.1 cm path-length cuvette. The DNA was complexed withpoly-L-lysine at identical molar ratios of amino to phosphate groups andvarious CD spectra compared: (A) standard control for DNA in 1 M NaCl;(B) Ψ-DNA as observed at a concentration of NaCl at which multimolecularaggregation occurs; (C) aggregated DNA shows turbidity and decreasedellipticity; (D) condensed, unimolecular complexes of DNA; and (E)relaxed DNA complex spectrum. The specta was taken at equalconcentrations of polymer and the signal for the buffer was subtractedin each case. Details of the assay are presented in the Methods.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The Multicellular Organism

Any multicellular organism into which it may be desirable to introduceexogenous nucleic acid is a potential subject for the present invention.The multicellular organism may be a plant or an animal, preferably thelatter. The animal is preferably a vertebrate animal, and morepreferably a higher vertebrate, i.e., a mammal or bird, the former beingespecially preferred. Among mammals, preferred subjects are human andother primates, laboratory animals such as mice, rats, rabbits andhamsters, pet animals such as dogs and cats, and farm animals such ashorses, cows, goats, pigs and sheep. It will be noted that these animalscome from four orders of class Mammalia: Primata, Rodenta, Carnivora andArtiodactyla.

The Target Cell

The target cells may belong to tissues (including organs) of theorganism, including cells belonging to (in the case of an animal) itsnervous system (e.g., the brain, spinal cord and peripheral nervouscells), the circulatory system (e.g., the heart, vascular tissue and redand white blood cells), the digestive system (e.g., the stomach andintestines), the respiratory system (e.g., the nose and the lungs), thereproductive system, the endocrine system (the liver, spleen, thyroids,parathyroids), the skin, the muscles, or the connective tissue.

Alternatively, the cells may be cancer cells derived from any organ ortissue of the target organism, or cells of a parasite or pathogeninfecting the organism, or virally infected cells of the organism.

A useful procedure for hepatic gene therapy requires an efficient andrelatively non-invasive approach to the introduction of genes ofinterest into the liver. Several techniques, employing receptor mediatedgene transfer, have been used with some success. However, there is aneed for a readily reproducible procedure which results in the prolongedexpression of the transgene in the liver, even in the absence of partialhepatectomy, and which therefore could be used for human gene therapy.Exogenous DNA has been introduced into hepatocytes of adult animals bytargeting the asialoglycoprotein (ASGP) receptor by means of aligand-poly-L-lysine biconjugate. For the ligand-targeting technique tobe efficient, the DNA must be in a form which permits it to remainintact in the blood and is small enough to be recognized by the ASGPreceptor on the surface of the hepatocytes. Wagner, et al. have targetedgenes to the transferrin receptor in hepatoma cells by condensing theDNA with a poly-L-lysine/transferrin conjugate, into a complex with adiameter of 80-100 nm. This size DNA conjugate is appropriate forrecognition by the transferrin receptor in hepatoma cells, but the ASGPreceptor of hepatocytes discriminates against ligands larger than 10-20nm in diameter.

The present inventors have developed a procedure for the introduction ofgenes into the liver of adult animals by receptor mediated uptake whichresulted in the expression of the gene for 140 days (the duration of theexperiment). This procedure has potential for application to human genetherapy. The major advantages of this method are: (i) the ease ofpreparation of the DNA complex; (ii) the ability to target genes tospecific tissues; (iii) the prolonged expression of the gene in theliver; (iv) the relative safety of the complex, since it is devoid ofinfectious viral DNA; and (v) the episomal maintenance of the introducedgene.

Targeting

A. Generally

"Targeting" is the administration of the compacted nucleic acid in sucha manner that it enters the target cells in amounts effective to achievethe clinical purpose. In this regard, it should be noted that DNA andRNA are capable of replication in the nucleus of the target cell, and inconsequence the ultimate level of the nucleic acid in the cell mayincrease after uptake. Moreover, if the clinical effect is mediated by aprotein expressed by the nucleic acid, it should be noted that thenucleic acid acts as a template, and thus high levels of proteinexpression can be achieved even if the number of copies of the nucleicacid in the cell is low. Nonetheless, it is desirable to compact highconcentrations of DNA to increase the number of target cells which takeup the DNA and the number of DNA molecules taken up by each cell.

The route and site of administration may be chosen to enhance targeting.For example, to target muscle cells, intramuscular injection into themuscles of interest would be a logical choice. Lung cells might betargeted by administering the compacted DNA in aerosol form. Thevascular endothelial cells could be targeted by coating a ballooncatheter with the compacted DNA and mechanically introducing the DNA.

In some instances, the nucleic acid binding moiety, which maintains thenucleic acid in the compacted state, may also serve as a targetingagent. Polymers of positively charged amino acids are known to act asnuclear localization signals (NLS) in many nuclear proteins. ApSV40-luciferase DNA condensed with poly-L-lysine, was injected in situinto the abdominal muscle of rats. Despite the absence of an explicittarget cell binding moiety, we observed a 20-fold higher luciferaseactivity in rats injected with the complexed DNA than in the ratinjected with naked DNA. Nonetheless, in some embodiments, targeting maybe improved if a target cell binding moiety is employed.

B. Use of a Target Cell Binding Moiety

If a TBM is used, it must bind specifically to an accessible structure(the "receptor") of the intended target cells. It is not necessary thatit be absolutely specific for those cells, however, it must besufficiently specific for the conjugate to be therapeutically effective.Preferably, its cross-reactivity with other cells is less than 10%, morepreferably less than 5%.

There is no absolute minimum affinity which the TBM must have for anaccessible structure of the target cell; however, the higher theaffinity, the better. Preferably, the affinity is at least 10³liters/mole, more preferably, at least 10⁶ liters/mole.

The TBM may be an antibody (or a specifically binding fragment of anantibody, such as an Fab, Fab, V_(M), V_(L) or CDR) which bindsspecifically to an epitope on the surface of the target cell. Methodsfor raising antibodies against cells, cell membranes, or isolated cellsurface antigens are known in the art.

The TBM may be a lectin, for which there is a cognate carbohydratestructure on the cell surface.

The target binding moiety may be a ligand which is specifically bound bya receptor carried by the target cells.

One class of ligands of interest are carbohydrates, especially mono- andoligosaccharides. Suitable ligands include galactose, lactose andmannose.

Another class of ligands of interest are peptides (which here includesproteins), such as insulin, epidermal growth factor(s), tumor necrosisfactor, prolactin, chorionic gonadotropin, FSH, LH, glucagon,lactoferrin, transferrin, apolipoprotein E, gp102 and albumin.

The following table lists preferred target binding moieties for variousclasses of target cells:

    ______________________________________                                        Target Cells                                                                              Target Binding Moiety                                             ______________________________________                                        liver cells galactose                                                         Kupffer cells                                                                             mannose                                                           macrophages mannose                                                           lung        Fab fragment vs. polymeric                                                    immunoglobulin receptor (Pig R)                                   adipose tissue,                                                                           insulin                                                           lymphocytes Fab fragment vs. CD4 or gp120                                     enterocyte  Vitamin B12                                                       muscle      insulin                                                           fibroblasts mannose-6-phosphate                                               nerve cells Apolipoprotein E                                                  ______________________________________                                    

Target binding moiety is not strictly necessary in the case of directinjection of the NABM/NA condensed complex. The target cell in this caseis passively accessible to the NABM/NA condensed complex by theinjection of the complex to the vicinity of the target cell.

C. Liposome-Mediated Gene Transfer

The possibility of detecting gene expresson by encapsulating DNA into aliposome (body contained by a lipid bilayer) using various lipid andsolvent conditions, and injecting the liposome into animal tissues, hasbeen demonstrated. However, despite the potential of this technique fora variety of biological systems, the DNA used in these experiments hasnot been modified or compacted to improve its survival in the cell, itsuptake into the nucleus or its rate of transcription in the nucleus ofthe target cells. Thus, these procedures have usually resulted in onlytransient expression of the gene carried by the liposome.

Cationic lipids have been successfully used to transfer DNA. Thecationic component of such lipids can compact DNA in solution. Thismethod has been shown to result in heavily aggregated DNA complexesthat, when used for transfecting the DNA in vitro, results in increasedefficiency of gene transfer and expression (relative to naked DNA).Although the formation of these complexes can promote gene transfer invitro, the injection of such complexes in vivo does not result in longlasting and efficient gene transfer.

The condensation procedure of the present invention provide structuralfeatures to the DNA/cationic lipid complex that will make it moreamenable to prolonged in vivo expression. The combination of suchmethods could be accomplished by either of two procedures:

1. Formation of condensed DNA complex that is later encapsulated usingneutral lipids into liposome bodies, or

2. Using the procedure described in this patent, the formation of highlycondensed unimolecular DNA complexes upon condensation with cationiclipids could be accomplished. These complexes should provide a higherefficiency of gene transfer into tissues of animals in vivo.

The procedure of the present invention for the condensation of DNA, ifcoupled to the encapsulation of the resulting compacted DNA into aliposome body, could provide a variety of advantages for transfectioninto animals:

1. The liposome promotes the passive fusion with the lipid bilayer ofthe cytoplasmic membrane of mammalian cells in tissues.

2. The condensed DNA could then transfer the genetic information with ahigher efficiency through the cell compartments to the nucleus for itsexpression.

3. Condensed DNA could be protected against degradation inside the cell,thus augmenting the duration of the expressio of the newly introducedgene.

4. Possible immunological response to the polycation condensed DNA couldbe avoided by the encapsulation with the immunologically inert lipidbilayer.

The Nucleic Acid Binding Moiety

Any substance which binds reversibly to a nucleic acid may serve as thenucleic acid binding moiety (NABM), provided that (1) it bindssufficiently strongly and specifically to the nucleic acid to retain ituntil the conjugate reaches and enters the target cell, and does not,through its binding, substantially damage or alter the nucleic acid and(2) it reduces the interactions between the nucleic acid and thesolvent, and thereby permits condensation to occur. The ultimatecriterion is one of therapeutic effectiveness of the conjugate.

Preferably, the NABM is a polycation. Its positively charged groups bindionically to the negatively charged DNA, and the resulting chargeneutralization reduces DNA-solvent interactions. A preferred polycationis polylysine. Other potential nucleic acid binding moieties includeArg-Lys mixed polymers, polyarginine, polyornithine, histones, avidin,and protamines.

The Nucleic Acid

Basic procedures for constructing recombinant DNA and RNA molecules inaccordance with the present invention are disclosed by Sambrook, J. etal., In: Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1989), which reference isherein incorporated by reference.

The nucleic acid may be a DNA, RNA, or a DNA or RNA derivative such as aderivative resistant to degradation in vivo, as discussed below. Withinthis specification, references to DNA apply, mutatis mutandis, to othernucleic acids as well, unless clearly forbidden by the context. Thenucleic acid may be single or double stranded. It is preferably of 10 to1,000,000 bases (or base pairs), more preferably 100 to 100,000, and thebases may be same or different. The bases may be the "normal" basesadenine (A), guanine (G), thymidine (T), cytosine (C) and uracil (U), orabnormal bases such as those listed in 37 CFR § 1.822 (p) (1). Thenucleic acid may be prepared by any desired procedure.

In a preferred embodiment, the nucleic acid comprises an expressiblegene which is functional in the target cell. For example, the gene mayencode coagulation factors, (such as Factor IX), enzymes involved inspecific metabolic defects, (such as urea cycle enzymes, especiallyornithine transcarbamylase, argininosuccinate synthase, and carbamylphosphate synthase); receptors, (e.g., LDL receptor); toxins; thymidinekinase to ablate specific cells or tissues; ion channels (e.g., chloridechannel of cystic fibrosis); membrane transporters (e.g., glucosetransporter); and cytoskeletal proteins, (e.g., dystrophin). The genemay be of synthetic, cDNA or genomic origin, or a combination thereof.The gene may be one which occurs in nature, a non-naturally occurringgene which nonetheless encodes a naturally occurring polypeptide, or agene which encodes a recognizable mutant of such a polypeptide. It mayalso encode an mRNA which will be "antisense" to a DNA found or an mRNAnormally transcribed in the host cell, but which antisense RNA is notitself translatable into a functional protein.

For the gene to be expressible, the coding sequence must be operablylinked to a promoter sequence functional in the target cell. Two DNAsequences (such as a promoter region sequence and a coding sequence) aresaid to be operably linked if the nature of the linkage between the twoDNA sequences does not (1) result in the introduction of a frame-shiftmutation in the region sequence to direct the transcription of thedesired gene sequence, or (3) interfere with the ability of the genesequence to be transcribed by the promoter region sequence. A promoterregion would be operably linked to a DNA sequence if the promoter werecapable of effecting transcription of that DNA sequence. In order to be"operably linked" it is not necessary that two sequences be immediatelyadjacent to one another. A nucleic acid molecule, such as DNA, is saidto be "capable of expressing" a mRNA if it contains nucleotide sequenceswhich contain transcriptional regulatory information and such sequencesare "operably linked" to nucleotide sequences which encode the RNA. Theprecise nature of the regulatory regions needed for gene expression mayvary from organism to organism, but in general include a promoter whichdirects the initiation of RNA transcription. Such regions may includethose 5'-non-coding sequences involved with initiation of transcriptionsuch as the TATA box.

If desired, the non-coding region 3' to the gene sequence coding for thedesired RNA product may be obtained. This region may be retained for itstranscriptional termination regulatory sequences, such as those whichprovide for termination and polyadenylation. Thus, by retaining the3'-region naturally contiguous to the coding sequence, thetranscriptional termination signals may be provided. Where thetranscriptional termination signals are not satisfactorily functional inthe expression host cell, then a 3' region functional in the host cellmay be substituted.

The promoter may be an "ubiquitous" promoter active in essentially allcells of the host organism, e.g., for mammals, the beta-actin promoter,or it may be a promoter whose expression is more or less specific to thetarget cells. Generally speaking, the latter is preferred. A promoternative to a gene which is naturally expressed in the target cell may beused for this purpose, e.g., the PEPCK (phosphoenol pyruvatecarboxykinase) promoter for expression in mammalian liver cells. Othersuitable promoters include albumin, metallothionein, surfactant, apoE,pyruvate kinase, LDL receptor HMG CoA reductase or any promoter whichhas been isolated, cloned and shown to have an appropriate pattern oftissue specific expression and regulation by factors (hormones, diet,heavy metals, etc.) required to control the transcription of the gene inthe target tissue. In addition, a broad variety of viral promoters canbe used; these include MMTV, SV-40 and CMV. An "expression vector" is avector which (due to the presence of appropriate transcriptional and/ortranslational control sequences) is capable of expressing a DNA (orcDNA) molecule which has been cloned into the vector and of therebyproducing an RNA or protein product. Expression of the cloned sequencesoccurs when the expression vector is introduced into an appropriate hostcell. If a prokaryotic expression vector is employed, then theappropriate host cell would be any prokaryotic cell capable ofexpressing the cloned sequences. Similarly, when a eukaryotic expressionvector is employed, then the appropriate host cell would be anyeukaryotic cell capable of expressing the cloned sequences.

In addition to or instead of an expressible gene, the nucleic acid maycomprise sequences homologous to genetic material of the target cell,whereby it may insert itself ("integrate") into the genome by homologousrecombination, thereby displacing a coding or control sequence of agene, or deleting a gene altogether.

In another embodiment, the nucleic acid molecule is "antisense" to agenomic or other DNA sequence of the target organism (including virusesand other pathogens) or to a messenger RNA transcribed in cells of theorganisms, which hybridizes sufficiently thereto to inhibit thetranscription of the target genomic DNA or the translation of the targetmessenger RNA. The efficiency of such hybridization is a function of thelength and structure of the hybridizing sequences. The longer thesequence and the closer the complementarily to perfection, the strongerthe interaction. As the number of base pair mismatches increases, thehybridization efficiency will fall off. Furthermore, the GC content ofthe packaging sequence DNA or the antisense RNA will also affect thehybridization efficiency due to the additional hydrogen bond present ina GC base pair compared to an AT (or AU) base pair. Thus, a targetsequence richer in GC content is preferable as a target.

It is desirable to avoid antisense sequences which would form secondarystructure due to intramolecular hybridization, since this would renderthe antisense nucleic acid less active or inactive for its intendedpurpose. One of ordinary skill in the art will readily appreciatewhether a sequence has a tendency to form a secondary structure.Secondary structures may be avoided by selecting a different targetsequence.

An oligonucleotide, between about 15 and about 100 bases in length andcomplementary to the target sequence may be synthesized from naturalmononucleosides or, alternatively, from mononucleosides havingsubstitutions at the non-bridging phosphorous bound oxygens. A preferredanalogue is a methylphosphonate analogue of the naturally occurringmononucleosides. More generally, the mononucleoside analogue is anyanalogue whose use results in oligonucleotides which have the advantagesof (a) an improved ability to diffuse through cell membranes and/or (b)resistance to nuclease digestion within the body of a subject (Miller,P. S. et al., Biochemistry 20:1874-1880 (1981)). Such nucleosideanalogues are well-known in the art. The nucleic acid molecule may be ananalogue of DNA or RNA. The present invention is not limited to use ofany particular DNA or RNA analogue, provided it is capable of fulfillingits therapeutic purpose, has adequate resistance to nucleases, andadequate bioavailability and cell take-up. DNA or RNA may be made moreresistant to in vivo degradation by enzymes, e.g., nucleases, bymodifying internucleoside linkages (e.g., methylphosphonates orphosphorothioates) or by incorporating modified nucleosides (e.g.,2'-O-methylribose or 1'-alpha-anomers). The entire nucleic acid moleculemay be formed of such modified linkages, or only certain portions, suchas the 5' and 3' ends, may be so affected, thereby providing resistanceto exonucleases.

Nucleic acid molecules suitable for use in the present invention thusinclude but are not limited to dideoxyribonucleoside methylphosphonates,see Mill, et al., Biochemistry, 18:5134-43 (1979), oligodeoxynucleotidephosphorothioates, see Matsukura, et al., Proc. Nat. Acad. Sci.,84:7706-10 (1987), oligodeoxynucleotides covalently linked to anintercalating agent, see Zerial, et al., Nucleic Acids Res., 15:9909-19(1987), oligodeoxynucleotide conjugated with poly(L-lysine), seeLeonetti, et al., Gene, 72:32-33 (1988), and carbamate-linked oligomersassembled from ribose-derived subunits, see Summerton, J., AntisenseNucleic Acids Conference, 37:44 (New York 1989).

Compaction of the Nucleic Acid

It is desirable that the complex of the nucleic acid and the nucleicacid binding moiety be compacted to a particle size which issufficiently small to achieve uptake by receptor mediated endocytosis,passive internalization, receptor-mediated membrane permeabilization, orother applicable mechanisms. Desirably, the complex of the compactednucleic acid, the target binding moiety, and the nucleic acid bindingmoiety is small, e.g., less than 100 nm, because the sinusoidalcapillary systems of the lung and spleen will trap aggregates of thatsize, and more preferably less than 80 or 90 nm, as that is the typicalinternal diameter of coated-pit endocytic vesicles. Since complexeslarger than 30 nm may be susceptible to nonspecific takeup bymacrophages in the spleen and liver, the conjugate is preferably alsosmaller than 30 nm.

In the case of the ASGP receptor of the liver, complexes larger than15-23 nm are excluded from uptake. This size limitation in vivo for thereceptor is probably directly related to the existence of anotherreceptor for galactosylated proteins in the Kupffer cells of the liver.The Kupffer cell receptor is very efficient in taking up and degradinggalactosylated molecules of larger size in vivo and thus, would competefor the uptake of the galactosylated DNA complex with the ASGP receptoron the surface of hepatocytes. Most preferably, for liver delivery, thecomplex is less than 23 nm, more preferably less than 15 nm, still morepreferably no more than 12 nm in diameter.

The present invention calls for the complex of the nucleic acid and thenucleic acid-binding carrier to be compacted without causing aggregationor precipitation, and preferably to a condensed state (see FIG. 12). Forthe purpose of the present invention, it is helpful to characterize DNAas having one of the following states: normal (uncondensed); condensed;relaxed; uni-aggregated (clusters of unimolecular toroids);multi-aggregated (clusters of multimolecular toroids); and precipitated.These states are defined in terms of their appearance under electronmicroscopy (see Table 103).

Condensed DNA is in a state in which interaction with the solvent isminimal and therefore the DNA is in the form of isolated spheres ortoroids. It is not fibrous to an appreciable degree. Relaxed DNA,typically formed by dissociation of polycation from the DNA, formsfibers. Aggregated DNA forms clumped or multimolecular toroids.

The theoretical size of a unimolecular DNA complex can be calculated bythe formulae set forth in legends "b" and "c" of Table 106. Preferably,the complexes of this invention have a diameter which is less thandouble the size calculated by one or both of these formulae. Largercomplexes are likely to correspond to multimolecularly aggregated DNA.

DNA can be compacted to a condensed state by neutralizing its charge,e.g., by addition of a polycation, or otherwise reducing itsinteractions with solvent. However, the polycation can cause aggregationor precipitation of the DNA if a chaotropic agent is not employed toprevent it. Compaction therefore can be accomplished by judicious use ofboth the polycation (to condense the DNA) and (as needed) of achaotropic agent (to prevent aggregation or precipitation).

Overuse of the chaotropic agent can, however, result in relaxation ofthe DNA. Preferably, the complex has a unaggregated, unimolecular toroidstructure condensed to smaller than 23 nm in diameter; the degree ofcompaction may be determined by electron microscopy. For example, acomplex of the PEPCK-hFIX gene with galactosylated polylysine has beencompacted to a unimolecular toroid with a mean diameter of about 12 nm,as shown in Table 106.

The term "unimolecular toroid" indicates that the toroid contains onlyone nucleic acid molecule; the toroid may contain many carrier (e.g.,galactosylated poly-Lys) molecules. A typical ratio is one DNA moleculeto about 100 carrier molecules, per "unimolecular" toroid.Alternatively, and perhaps more precisely, this structure may bereferred to as a mono-nucleic acid toroid. Unimolecular andmultimolecular toroids (the latter each contain more than one DNAmolecule) may be distinguished by the different size of each of thecomplexes when viewed by the electron microscope, indicating the multi-or unimolecular (counting only the DNA molecules) composition of thetoroids.

We have also used other techniques to identify structural changes in theDNA upon poly-L-lysine binding. The first of these is thespectrophotometric determination of the turbidity in the solution usingthe absorbance at 400 nm. Turbidity is primarily an indicator ofaggregation. Aggregation is confirmed by a circular dichroism (CD) valuegreater than 0 at wavelengths from 300 to 340 nm.

FIG. 18 illustrates the effect on turbidity of adding the poly-L-lysineto the DNA solution at different starting concentrations of NaCl.Turbidity increases as the initial concentration of salt is increased(this could be easily confirmed by eye), indicating that thecondensation of the DNA complex at lower ionic strength results in asuspension of particles composed of unimolecular DNA-poly-L-lysinecomplexes interacting with each other. We noted that the solutions ofDNA condensed at lower salt concentration were clear, with the presenceof particulate matter in suspension. Solutions containing the DNAcomplex with different degrees of turbidity were analyzed by EM tovisualize the DNA structures formed in each situation. Appropriatelycondensed, unimolecular DNA complexes were found with both clear andslightly turbid solutions. This was not true for the condensation of DNAcomplexes at initial low ionic strength where we noted minimalabsorbance at 400 nm (FIG. 18) because the solutions containingparticles in suspension did not absorb at 400 nm. However, when thesesolutions were analyzed using EM, we noted the expected transitionalstructures shown in FIG. 1. When the particles in suspension becametotally dispersed, the structures identified by EM were essentiallyidentical to condensed unimolecular DNA complexes. Thus, turbidity ofthe solution containing the DNA complexes is dependent on the initialconcentration of salt used for condensation of the complex. Although themechanisms responsible for the observed differences in the condensationof DNA at initial low and high ionic strength is not clear, we adaptedour protocol to appropriately condense DNA, avoiding the formation ofturbid solutions.

A more reliable technique for diagnosing the structural transition ofDNA-poly-L-lysine complexes in solution is the absorbance of thecondensing complex at 260 nm as the concentration of NaCl increases. Theuni-aggregated DNA complex in suspension has only 10-30% of the expectedabsorbance because the particulate matter does not absorb at 260 nm. Theaddition of NaCl disperses the uni-aggregated DNA complex in suspensionwhich results in the observed steep increase in the absorbance noted inFIG. 11. At this point the solution is clear and there are no visibleparticulate structures in suspension. This feature of theDNA-poly-L-lysine condensation clearly correlates with the structuresshown in FIG. 1. At a concentration of NaCl which causes a steepincrease in the absorbance at 260 nm, we observed unaggregated,condensed complexes by EM; before this critical concentration of NaClwas attained, the DNA complex appear aggregated and at higher NaClconcentrations the DNA complex was relaxed. A second transition inabsorbance at 260 nm, as a result of the relaxation of the condensed DNAcomplex that was in suspension, indicates the full solubilization of theDNA complex.

Circular dichroism (CD) can be used to monitor the condensation of DNA.When the spectrum is identical to that of DNA alone, then the DNAcomplex is assumed to be correctly compacted, i.e., into unimolecularcomplexes. In another words, the positive spectrum at 220 nm isquantitatively similar to the 220 nm spectrum of DNA alone, and thecross-over (the wavelength at which the spectrum of the complex crossesthe 0 point) is essentially identical to that of DNA alone. When the DNAaggregates into multimolecular complexes, the positive spectrum at 270nm is inverted into a negative spectrum at that wavelength (this iscalled psi-DNA structure or ψ-DNA).

Table 103 sets forth the characteristics of each state as determined bynaked eye observation, circular dichroism spectroscopy, electronmicroscopy, and absorbance at 260 nm.

It should be noted that any other techniques which are capable ofidentifying condensed DNA complexes may be used instead of or incombination with those discussed above.

To compact the nucleic acid, the carrier is added to the nucleic acidsolution, whereby the carrier disrupts the nucleic acid: solventinteractions allowing the nucleic acid to condense. Preferably, at leastthe turbidity of the solution is monitored as the carrier is added, sothat a change in state is promptly detected. Once turbidity appears, thestate of the DNA may be further analyzed by CD spectroscopy to determinewhether the DNA is in the condensed or the aggregated state.(Precipitation should also be detectable with the naked eye.)Preferably, the carrier is added sufficiently slowly to the nucleic acidsolution so that precipitation and aggregation are minimized. Ifprecipitation or aggregation occur, a chaotropic salt should be addedslowly, and the result again examined by CD spectroscopy. The preferredsalt is NaCl. Other chaotropic salts can be used as long as they aretolerated by the animal (or cells) to which they will be administered.Suitable agents include Sodium sulfate (Na₂ SO₄), Lithium sulfate (Li₂SO₄), Ammonium sulfate ((NH₄)₂ SO₄, Potassium sulfate (K₂ SO₄),Magnesium sulfate (MgSO₄), Potassium phosphate (KH₂ PO₄), Sodiumphosphate (NaH₂ PO₄), Ammonium phosphate (NH₄ H₂ PO₄), Magnesiumphosphate (MgHPO₄), Magnesium chloride (MgCl₂), Lithium chloride (LiCl),Sodium chloride (NaCl), Potassium chloride (KCl), Cesium chloride(CaCl), Ammonium acetate, Potassium acetate, Sodium acetate, Sodiumfluoride (NaF), Potassium fluoride (KF), Tetramethyl ammonium chloride(TMA-Cl), Tetrabutylammonium chloride (TBA-Cl), Triethylammoniymchloride (TEA-Cl), and Methyltriethylammonium chloride (MTEA-Cl)

We have investigated variables that affect condensation of DNA in vitroand the functional relevance of these parameters for efficient deliveryof DNA complexes into animals by receptor-mediated endocytosis. We noteda strong correlation between the ionic strength at which the condensedDNA-poly-L-lysine complex remains stable in solution and theconcentration of DNA. These experiments were performed using a 4.5 kbplasmid containing the promoter from the gene for PEPCK linked to thestructural gene for hFIX, using a ratio of DNA to poly-L-lysine thatresulted in a 1 to 1 ratio of negative to positive charges in solution.The variation in the final concentration of NaCl necessary to solubilizethe particles is a logarithmic function of DNA concentration, in whichthe condensation of highly concentrated DNA-poly-L-lysine complexesoccurs with only a slight increase in ionic strength. This physicalcharacteristic of DNA condensation has clear advantages for the deliveryof the DNA particles to tissues of adult animals in vivo since it haslittle effect on the ionic load in the animal's blood.

The linear fit of the data using the least square method is described bythe following function:

    log.sub.10 (NaCl, mM)=b0*(DNA, μM Phosphate)+b1r2=0.97 where b0=2.52×10 E-3, b1=0.577

We have observed variations in the function described by the aboveequation when we use different DNA plasmids and different DNApreparations during the condensation process. These differences areprobably related to the variation in the affinity of poly-L-lysine forDNA of different sources and compositions. For maximum binding affinitywe generally use DNA precipitated twice with sodium acetate and 2.5volumes of -40° C. ethanol (see Methods). We have not found an apparentdifference in binding affinity of poly-L-lysine for DNA of differentforms (i.e., supercoiled, nicked and linear) and for DNA extracted usinganionic exchange chromatography or cesium chloride gradientcentrifugation. This may indicate the presence of a contaminant in theDNA preparations from different sources which has poly-L-lysine bindingactivity, that is eliminated by sequential DNA precipitation.

We have also investigated the effect of the length of the poly-L-lysineon the concentration of NaCl necessary for the effective condensation ofDNA (FIG. 19). The correlation between these variables was assessedusing a fixed concentration of DNA from different sources. We have useda broad range of poly-L-lysine lengths; essentially the sizes ofpoly-L-lysine available commercially. However, the length of thepoly-L-lysine in an average of various sizes of the protein asdetermined by low-angle light scattering analysis of individual lots ofchemically synthesized poly-L-lysine. The actual distribution of sizeswithin each sample varies from 60 to 80% of the material beingdistributed, which is ±20% from the average size. This broaddistribution within a single size is a source of error in ourdeterminations. Nevertheless, there is a clear correlation observable inFIG. 19 between the length of the poly-L-lysine and the necessaryconcentration of NaCl needed for the condensation of the DNA complex insolution. This correlation is a linear function of poly-L-lysine lengthup to a size of 150 lysine residues, after which the function reachessaturation and there is no increase in the concentration of NaCl neededfor the condensation of DNA with longer poly-L-lysine. These data areconsistent with a cooperative binding between the poly-L-lysine and theDNA phosphate backbone. Thus, by reducing the length of thepoly-L-lysine molecules used to condensed the DNA the solution of DNAcomplex injected into the animals will be less hypertonic. It is alsoimportant to consider the dilution of the DNA complex in the blood ofthe animal to evaluate the functional significance of these changes inionic strength on the efficiency of this method for gene therapy. Wehave injected rats with DNA complexes containing longer range ofpoly-L-lysine lengths than those shown in FIG. 19 and rabbits with theshorter range of sizes of poly-L-lysine, and noted positive andpersistent expression of the transfected genes in both cases.

The preferred minimum initial salt concentration is dependent on thecompaction activity of the carrier and the chaotropic activity of thesalt. If the NABM were (Lys)₈, or (Lys)₂₇, the initial NaClconcentration could be zero. With longer polyLys chains, however, in theabsence of NaCl, precipitation would be immediate. With (Lys)₅₀, theinitial NaCl concentration is preferably be at least about 300 mM.Nonetheless, if the TBM is a protein that affects the condensation, theinitial salt concentration could be as low as zero.

The carrier may be added continuously, or in small discrete steps. Onemay begin with a higher flow rate, or larger aliquots, and reduce theflow rate or aliquot size as the desired endpoint of the reaction isneared. Typically 0.1 to 10% of the carrier solution is added at a timeto the DNA solution. Each addition is preferably made every 2 seconds to2 minutes, with constant vortexing. However, longer settlement times maybe allowed.

In one embodiment, a nucleic acid, contained in a salt solution, whichis preferably at least 0.5 M, but less than 1.5 M NaCl, is mixed withpoly-L-lysine (109 lysines) containing the covalently linked target cellbinding moiety (for example, galactose), which is contained in asolution of NaCl at the same concentration (e.g., 0.5 to 1.5 M NaCl).Preferably, the molar ratio of nucleic acid phosphate group topositively charged group of the DNA binding moiety is in the range of4:1 to 1:4, and more preferably is about 1.5:1.

Some of Applicants' experimental results are set forth in Table 104. Wehave taken 16 examples (asterisked in the first column of Table 104)which were tested and worked in vivo, and regressed final NaClconcentration (the independent variable) against DNA concentration andpoly-L-Lys length (the dependent variables), with the results given inTable 105.

The Conjugation

In the embodiments relying on a target-binding carrier molecule, thenucleic acid binding moiety will be conjugated, covalently ornoncovalently, directly or indirectly, to the target cell bindingmoiety. The conjugation may be performed after, or, more usually before,the loading of the nucleic acid binding moiety with the nucleic acid ofinterest. Either way, the conjugation should not substantially interferewith the binding of the nucleic acid to the nucleic acid binding moiety,or, for that matter, with the ability of the target cell binding moietyto bind to the target cell.

Pharmaceutical Compositions and Methods

The compacted nucleic acid, optionally conjugated with a TBM, may beadmixed with a pharmaceutically acceptable carrier for administration toa human or other animal subject. It will be appreciated that it ispossible for a DNA solution to contain both condensed DNA and relaxedDNA. The compositions of this invention preferably are sufficiently richin condensed complexes so that the absorbance at 260 nm is less than 50%that of naked DNA of equal concentration. As stated in Table 103,condensed DNA usually has an absorbance of 20-30%, and relaxed DNA,80-100%, that of naked DNA.

The administration may be by any suitable route of administration. Thedosage form must be appropriate for that route. Suitable routes ofadministration and dosage forms include intravascular (injectablesolution), subcutaneous (injectable solution, slow-release implant),topical (ointment, salve, cream), and oral (solution, tablet, capsule).With some routes of administration, the dosage form must be formulatedto protect the conjugate from degradation, e.g., by inclusion of aprotective coating or of a nuclease inhibitor.

The dosage may be determined by systematic testing of alternative doses,as is conventional in the art.

Rats (200-300 g) tolerate as much as 600 μg doses of the DNA complex ofExample 1 without any apparent ill effects on growth or health. Mice (25g) have been injected with 150 μg of that DNA complex without anyapparent problem.

In humans, a typical trial dose would be 60-120 mg of DNA; if this doseis too low to be effective or so high as to be toxic, it may beincreased, or decreased, respectively, in a systematic manner, until asuitable dose is identified.

For short life span cells, e.g., macrophages, a typical dosing schedulemight be one dose every two weeks. For long life span cells, e.g.,hepatocytes, one dose every two months might be preferable.

Adjuvants may be used to decrease the size of the DNA complex (e.g.,2-10 mM MgCl), to increase its stability (e.g., sucrose, dextrose,glycerol), or to improve delivery efficiency (e.g., lysosomotropicagents such as chloroquine and monensine). The complexes may be enclosedin a liposome to protect them and to facilitate their entry into thetarget cell (by fusion of the liposome with the cell membrane).

The invention is illustrated, but not limited, by the followingexamples.

EXAMPLE 1 Introduction

Christmas disease, or Hemophilia B, is a sex-linked recessive bleedingdisorder due to a deficiency of functional coagulation factor IX in thecirculation. Human factor IX (hFIX) is a plasma glycoprotein normallysynthesized in the liver, that plays an integral role in the intrinsiccoagulation pathway. Once it has been converted to its serine proteaseform (IXa) by activated plasma thromboplastin antecedent (factor XIa),the activated protein interacts with coagulation factor VIIIa, calciumions, and phospholipids to produce a complex that converts factor X toXa. Factor IX undergoes several post-translational modifications in theliver that are essential for its function before secretion into theblood. These include Vitamin K dependent γ-carboxylation ofamino-terminal glutamic acid residues and β-hydroxylation of asparticacid.

Christmas disease accounts for approximately 10 to 20 percent of allinherited clotting disorders. Affected individuals exhibit a wide rangeof clinical severity that generally correlates with the level ofcirculating factor IX. Patients with severe deficiencies of functionalfactor IX may bleed spontaneously into soft tissues and joints or afterminor trauma. Transfusions of plasma or concentrates rich in factor IXare used to abort bleeding episodes by temporarily correcting thedeficiency. Unfortunately, clinical management has been confounded byviral contamination of pooled plasma. Blood-borne infections, such ashepatitis and the acquired immunodeficiency syndrome, have becomesignificant problems in the treatment of the hereditary clottingdisorders. These complications stress the importance of developingalternative treatments.

The gene for human coagulation factor IX has been identified andsequenced; 1,248 base pairs, in length, the complementary DNA predicts aprotein of 416 amino acids, and, after post-translational modifications,the mature protein has a molecular weight of approximately 54,000 Da. Agene encoding human coagulation factor IX may be used for geneticcorrection of hemophilia B.

A chimeric P-enolpyruvate carboxykinase-human factor IX(PEPCK-hFIX) gene(50% supercoiled/50% open circular) was condensed with galactosylatedpoly-L-lysine (average length 50 or 109 amino acids) by titration withNaCl. This process was monitored using CD spectroscopy and electronmicroscopy and resulted in the formation of a DNA-carrier complex of10-12 nm in diameter at a critical NaCl concentration. We haveintroduced the PEPCK-hFIX gene, conjugated using this procedure, intothe intact livers of adult rats and have demonstrated that theDNA-carrier complex specifically targets the gene to this organ and thathFIX DNA, mRNA and hFIX protein can be demonstrated up to 140 days (theduration of the experiment) after administration of the DNA-carriercomplex. The gene is present as an episome as determined by Southernanalysis of DNA isolated from the liver of an animal 32 days afterinjection of the DNA-conjugate. Transcription of the PEPCK-hFIX gene wascontrolled by diet for the entire time course of the experiment; feedingthe animals a carbohydrate-free diet for one week resulted in thepredicted induction of hFIX in the blood, as detected by Western blothybridization.

Methods

A. Galactosylation

Polymers of L-lysine-HBr or L-lysine-Cl with an average chain length of109 (Sigma) were galactosylated essentially as described by Monsigny, etal. (1984) Biol. Cell., 51, 187. Briefly, 2 mg of poly-L-lysine wasreacted with 89 g of α-D-galactopyranosyl phenyl-isothiocyanate (SigmaG-3266) dissolved in N,N-Dimethyl formamide (5 mg/ml). The solution wasadjusted to pH 9.0 by the addition of 1/10 volume of 1 M sodiumcarbonate pH 9.0. Since the reaction is 10% efficient, 0.8% of the ε-NH3groups present in the solution are glycosylated. The tube was shieldedfrom light by aluminum foil and mixed for 6 hours at room temperature.The solution was then dialyzed, using Spectra-Por dialysis tubing(Fisher 3500 M.W. cutoff), against 500 ml of 5 mM NaCl buffer for 2 dayswith frequent changes of buffer (2 changes/day).

B. Analysis of the Ligand

The dialyzed solution was then analyzed spectrophotometrically at 205 Åand 250 Å for the concentration of poly-L-lysine and the concentrationof phenyl-galactose residues, respectively. This step ensures thatsignificant losses during dialysis have not occurred, and that thegalactosylation reaction was complete, since in the solution only themodified galactose will absorb at 250 Å.

C. Complex Formation

Plasmid DNA was prepared using standard techniques. The DNA wasresuspended in 10 mM Tris-HCl, pH 8.0, containing 1 mM EDTA and theconcentration of the DNA determined spectrophotometrically. The DNApreparation was digested twice with RNAses A+T1. This step ensures thatRNA is not present in the solution (RNA inhibits the condensation of DNAby poly-L-lysine). A solution containing a high concentration of DNA(1.5-2 mg/ml) was used in further steps. An example of a typicalprotocol for DNA condensation is described as follows:

a) 300 μg of DNA in 200 μl of 0.75 M NaCl (added from 5 M NaCl solution)is vortexed at medium speed, using a VIBRAX machine (IKA-VIBRAX-VXR).This procedure is desirable to increase the effective length of the DNApolymer in high salt solutions, thus achieving efficient binding of thepoly-L-lysine moiety to the DNA backbone.

b) 84 μg of poly-L-lysine-galactose in 200 μl of 0.75 M NaCl (added froma 5 M NaCl solution) is added dropwise over a period of 30 minutes to 1hour in 20 μl aliquots. This amount translates into a molar ratio of 1DNA PO₄ ⁻ group to 0.7 carrier NH₃ ⁺ groups.

c) The solution becomes turbid at the end of the process. 3 μl aliquotsof 5 M NaCl are added dropwise to the vortexing solution until turbiditydisappears as monitored by eye. This process is slow, allowing 30seconds between the addition of each new aliquot of 5 M NaCl. Then thesolution is subjected to CD spectroscopic monitoring while 2 μl aliquotsof 5 M NaCl are gradually added. The condensation process is completewhen the diagnostic spectrum of the DNA complex is observed. Forsubsequent preparations of DNA complex consisting in the same plasmidDNA at the same concentration of nucleotide, the protocol can befollowed without monitoring with CD and the results will be fullyreproducible. When using different concentration of DNA or a differentplasmid the CD monitoring should be repeated.

We have found that an alternative technique for monitoring DNA complexformation gives similar results. This technique consists of thefollowing steps:

a) and b) Idem.

c) The solution becomes turbid at the end of the process. 3 μl aliquotsof 5 M NaCl are added dropwise to the vortexing solution until turbiditydisappears as monitored by eye. This process is slow, allowing 30seconds between the addition of each new aliquot of 5 M NaCl. Thesolution is then centrifuged at full speed (12000×g) for 30 secondsusing a microcentrifuge and the appearance of precipitate is monitored.If a precipitate is observed 2 μl aliquots of 5 M NaCl are added. Thesolution is further vortexed for 0.5 minutes and the centrifugation stepis repeated. The appearance of a precipitate is due to the aggregationof the DNA-complex in solution and indicates that the DNA has not beenfully collapsed.

Results and Discussion

In developing the procedure described herein, we have monitored thephysical structure of the DNA/ligand-poly-L-lysine conjugate usingcircular dichroism (CD) and electron microscopy and studied theconditions by which a functional complex is generated. We thendetermined the functional relevance of the physical structure of theDNA/ligand-poly-L-lysine conjugate using intact animals. The DNA wascondensed by the addition of the ligand-poly-L-lysine in the presence ofvarying concentrations of NaCl. Either 60 μg of RNA-freeCMV-β-galactosidase (A) or phFIX (B,C,D, and E), diluted to a finalvolume of 150 μl in 700 mM NaCl were vortexed at medium speed in aVIBRAX apparatus (IKA-VIBRAX-VXR). 19 μg of α-galactopyranosyl-phenylisothiocyanate/poly-L-lysine biconjugate (Sigma) were diluted in thesame way and added dropwise to the vortexing solution of DNA. For invivo studies, 300 μg of DNA (dissolved in TE buffer, pH 8) in 150 μl of700 mM NaCl were condensed with 95 μg of α-galactopyranosyl-phenylisothiocyanate/poly-L-lysine biconjugate in 150 μl of 700 mM NaCl. Theslow addition of the polycation results in the formation of a turbidsolution which is dissolved by the stepwise addition of 3 μl aliquots of5M NaCl. The disappearance of the turbidity was monitored by eye and atthe point of no turbidity the solutions of DNA/poly-L-lysine complexeswere investigated by both electron microscopy (E.M.) and CDspectroscopy.

Continuing addition of 2 μl aliquots of 5M NaCl resulted in structuralchanges as shown in FIGS. 1A-1F. Representative spectra demonstratingdifferent structural conformations of the DNA complex at variousconcentrations of NaCl and in the presence and absence of addedpoly-L-lysine, are presented in FIG. 1. Polycation binding to DNAresults in a specific spectrum characterized by a displacement of thecross-over to longer wavelengths; this shift can be correlated with thechiral packing of DNA/poly-L-lysine conjugates in high order, asymmetricstructures similar to the Y-form of DNA. As shown in FIG. 1A, doublestranded DNA (in 1M NaCl) has a characteristic spectrum which wasmarkedly altered by the addition of poly-L-lysine at varying ionicstrengths. (FIG. 1A). When the ionic strength of theDNA/ligand-poly-L-lysine conjugate was increased the complex proceededthrough a transition from an aggregated (FIG. 1C) to a condensed state(FIG. 1D & FIG. 1E). This corresponds to a shift in the spectrum of thecomplex as shown in FIG. 1A. The change in the CD spectra at 220 nm andthe shift in the cross-over (0 line in FIG. 1A) that occurs withincreasing ionic strength of the solution is of particular importance inmonitoring the formation of condensed DNA complex by means of CDspectroscopy. If the ionic strength is increased above the criticalrange required for the condensation of the DNA complex, the complexassumes a non-condensed, relaxed conformation (FIG. 1F). This transitionin the conformation of the DNA complex cannot be monitored by CDspectroscopy so that a rigorous titration of NaCl is critical to thesuccess of this procedure. It is important to note that the diameter ofthe DNA complex observed in FIG. 1D (about 10 nm) conforms with thediscrimination range desirable for internalization of molecular ligandsby the hepatic receptor for asialoglycoproteins.

We therefore verified the functional relevance of the observed DNAstructures as vehicles to transfer of the DNA into hepatocytes in vivoby receptor-mediated endocytosis. In order to establish the nature ofthe uptake process, we followed the removal of the DNA complex from themedia by HepG2 cells, which contain the asialoglycoprotein receptor. Theuptake of the DNA complex was completely inhibited when a 100-fold molarexcess asialogetuin was used as a competitor, indicating that thecomplex was being taken up by receptor-mediated endocytosis via theASGP.

A plasmid (pPFIX) containing a chimeric gene composed of the promoter ofthe gene for the cytosolic form of P-enolpyruvate carboxydinase (PEPCK)from the rat, linked to the cDNA for human coagulation Factor IX (hFIX)(Ferkol, et al., FASEB J, 7:1081 (1993)) was used to follow the deliveryand expression of the DNA in the liver. The time-course of expression ofhFIX gene in the transfected animals was determined by Western blothybridization, using a monoclonal antibody against the mature hFIXpeptide.

Adult, male Sprague-Dawley rats, approximately 250 g in weight, wereanesthetized with ether. 300-400 μl of a solution containing 300 μg ofpPFIX complexed as previously described with galactose-poly-L-lysine,were infused into the caudal cava vein. Rats were killed at 0, 4, 8, 12,32, 72 and 136 days after transfection and tissues and blood samplestaken.

Plasma samples (1 μl) from transfected animals and a 1:4 dilution of ahuman plasma control were subjected to electrophoresis in SDS/10%polyacrylamide gels and transferred onto nitrocellulose membrane filtersusing standard techniques. The blots were block with 1× PBS, pH 7.4,0.03% polyoxyethylene sorbitan monolaurate (Tween 20), and 10% (w/v) dryskim milk for two hours at room temperature, followed by incubation witha 1/1000 dilution of a monoclonal murine anti-human factor IX antibody(3 μg/ml) for two hours at room temperature. The monoclonal antibody waskindly provided by Dr. Kenneth Smith (United Blood Services,Albuquerque, N. Mex.). The membrane was washed three times in 1× PBS, pH7.4 and 0.03% Tween 20, then incubated with a 1/500 dilution of goatanti-murine lgg (H+L)--horseradish peroxidase conjugate. The membranewas then washed vigorously four times with 1× PBS, pH 7.4 and 0.03%Tween 20, and 10 ml of Western blot enhanced chemiluminescence detectionsolution was applied for one minute. The luminescence emitted from thefilter was detected by a 20 second exposure to photographic film. Wedetected a band hybridizing specifically to the hFIX monoclonal antibodyfor as long as 140 days. No hybridizing band was detected inuntransfected controls.

The liver from an animal 32 days after transfection was taken andgenomic DNA isolated using standard techniques. 5 μg of total DNA fromthe transfected animal and from a non-transfected control were digestedwith either EcoRI or BgI II overnight. Southern blot electrophoresis wasperformed by established methods. The DNA from the transfected animalonly hybridized to 4.5 kb BglII and a 2.6 kb EcoRI probes.

Spleen, lung, heart and liver tissues were obtained from a rattransfected with 300 μg of the DNA complex. PCR analysis was carried outon total genomic DNA isolated from these tissues. Only the liver of thetransfected rat, and not its spleen, lung or heart, or the liver of acontrol animal, was positive for the 720 bp probe.

The presence of mRNA transcripts for human factor IX in the livers ofrats transfected with pFIX was determined after treatment of totalcellular hepatic RNA with Moloney Murine Leukemia virus reversetranscriptase and amplification of the resultant cDNA by the polymerasechain reaction. Briefly, 1 μg of total rat liver RNA was treated with 10U DNAse I (RNAse free), and added to a solution containing 500 nM of(dT)₁₆ oligonucleotide primer and 500 nM of each dNTP, and heated to 42°C., and 1 μl of the cDNA pool was amplified by the polymerase chainreaction, using primers expanding the 5' UTR region of the PEPCKpromoter and the cDNA for hFIX. As a control, the same RNA samples notconverted to cDNA by reverse transcriptase were also used as polymerasechain reaction templates to ensure that contaminating plasmid DNA hadnot been amplified. The products were separated by agarose gelelectrophoresis and Southern blot hybridization using a radiolabeledhuman factor IX cDNA probe. We observed a band that hybridizedspecifically with the hFIX probe only in the transfected animals. Nobands were detected in either non-transfected controls or transfectedsamples not converted to cDNA by reverse transcriptase.

The functional activity of hFIX in the plasma of transfected animals wasanalyzed by measuring the procoagulant activity of human Factor IX. Amodification of the one stage, kaolin-activated, partial thromboplastintime with factor IX-deficient human plasma was used. Blood samples wereobtained from experimental animals by venipuncture. One fiftieth volumeof 500 mM sodium citrate pH 5.0, was added to prevent coagulation, andthe plasma was stored at 20° C. The samples were assayed in duplicate,and their activity ws compared to the functional activity of pooledplasma from 24 normal adult human males. In normal human plasma isequivalent 100% functional activity or approximately 3 μg of humanFactor IX per ml. Background Factor IX activity in rat plasm(approximately 0.15 units/ml of Factor IX activity in rat serum) wassubtracted from each value of human Factor IX determined in individualanimals. The background values is non-specific cross activity of ratFactor IX determined in the human Factor IX assay used in this analysis.Blood samples were obtained from experimental animals by venipuncture.One fiftieth volume of 500 mM sodium nitrate, pH 5.0, was added toprevent coagulation, and the plasma was stored at 20° C. The normalconcentration of hFIX in human plasma is 3 μg/ml, Approximately 15 ng/ml(72 days after transfection) to 1050 ng/ml (48 days after transfection)of active human factor IX were produced in individual animals injectedwith the DNA complex (Table 102). It is not clear if the smallvariations in the concentration of recombinant hFIX found in the animalsrepresent a difference in delivery efficiency or in the expression ofthe newly introduced gene. The hFIX gene was expressed in the animalsfor up to 140 days (the duration of the experiment), with the highestlevel noted at 48 days (Table 102).

It has been established using transgenic animals (McGrane, et al., 1988,1990; Short, et al. 1992) that transcription from the PEPCK promoter canbe induced by the administration of a high protein-low carbohydratediet. In order to demonstrate the regulated expression of the transgene,we analyzed the blood of transfected animals for the presence of hFIX byWestern blot hybridization before and after feeding a high protein-lowcarbohydrate or a normal chow diet for 1 week. We noted up to 3-foldinduction of PFIX gene expression in animals containing the PFIX genefor up to 140 days after injection of the DNA complex. The samePEPCK-hFIX gene, introduced into the livers of rats using an alternativemethod of receptor-mediated gene transfer targeting the ASGE, was activefor only two days (Ferkol, et al., 1993); this suggests that the use ofa highly compacted DNA complex may be responsible for the prolongedexpression of the transgene noted in the present study.

Detection of maintained levels of hFIX protein at time points as long as140 days is evidence for expression throughout the experimental timecourse. A human FIX 800 bp. specific transcript was detected by PCRamplification of cDNA generated from total cellular RNA by reversetranscriptase, in the livers of animals expressing functional hFIXprotein (FIG. 3A). The presence of mRNA along the experimentaltime-course would indicate that there is a maintained pool oftranscriptionally active DNA in these animals which persistence willexplain the prolonged expression and detection of hFIX and specificmRNA.

We have also established the presence of the transfected DNA in theliver of animals 32 days after transfection, and investigated itsphysical state. The DNA extracted was subjected to restriction enzymeanalysis with BglII that linearizes the plasmid (4.5.Kb) and with EcoRIthat releases the 2.6 Kb chimeric gene from the plasmid. Southern blothybridization using a hFIX specific probe demonstrated that thetransfected DNA remains in episomal state in the transfected livers,since BglII produced a single band consistent with the size of thelinear plasmid in contrast to the expected smeared hybridization whenrandom integration occurs (FIG. 3B). We cannot rule out the possibilitythat a small proportion of the transfected DNA may have undergone randomintegration into the genome of the transfected animals. However, webelieve that this event is improbable since the liver has not beensubjected to stimulation of mitosis (i.e., partial hepatectomy).

The asialoglycoprotein receptor is present only in parenchymal cells ofthe liver. Nevertheless, it has been shown that asialoglycoproteins andother galactose terminal ligands can be taken up by macrophages by amechanism dependent on the size of the molecular ligand. SeeSchlepper-Schafer, J. et al., Exp. Cell. Res. 165:494 (1986);Bijsterbosch, M. K., et al., Mol. Pharmacol 36:484 (1989); andBijsterbosch, M. K., et al., Mol. Pharmacol 41:404 (1992). The size ofthe DNA/ligand-poly-L-lysine complex in our experiments would becompatible with the discriminating range of the asialoglycoproteinreceptor. In order to investigate the specificity of the DNA complex wehave obtained DNA from different tissues in a transfected animal andamplified the transfected DNA by PCR. Our results show the absence ofamplifiable DNA in tissues other than liver, which would indicatespecific uptake by hepatocytes. It is especially interesting that thereis no detectable uptake in macrophage-containing tissues like lung andspleen. In contrast, we have detected transfected DNA in the lung andspleen of animals transfected using the method described by Wu, et al.for receptor-mediated endocytosis by means of the asialoglycoproteinreceptor. We believe that the small size of the molecular ligandachieved in our experiments is responsible for the specificity of uptakereported here.

EXAMPLE 2

In this Example a different promoter-gene construct (SV40/luciferase) isdelivered to a different cell type (macrophages) by means of a differenttarget cell binding moiety.

Introduction

The recognition and uptake of circulating glycoproteins by specificcells are determined by the nature of the exposed sugar residues presenton the surface of the molecule. The clearance systems of specificglycoproteins are relatively exclusive and are mediated by specifictypes of cells. The mannose receptor recognizes glycoproteins withmannose, glucose, fucose, and N-acetylglucosamine residues in exposed,non-reducing positions. Various proteins and glycoprotein conjugatesbearing these carbohydrate residues bind to isolated alveolarmacrophages, and mannose-terminal glycoproteins infused into thecirculation of rats are cleared by Kupffer cells in vivo. Conversely,galactose-terminal glycoproteins, which are cleared by theasialoglycoprotein receptor on hepatocytes, are not recognized by thesecells. This cell-surface receptor is expressed by a variety ofmacrophage subtypes but not circulating monocytes, and mediates thedelivery and internalization of mannose-terminal glycoproteins. Themannose receptor recycles constituitively from a pre-lysosomalcompartment to the cell surface, and receptor expression is regulated bymacrophages.

Macrophages present in various organs (i.e., liver, spleen, lung, andbone marrow) which bind mannose-terminal glycoproteins and therefore maybe a target cell for receptor-mediated gene transfer. We tested thishypothesis by examining our ability to deliver functional exogenousgenes cells which express the mannose receptor. In this report, amannose-terminal neoglycoprotein carrier was synthesized and employed asa ligand for receptor-mediated gene transfer to primary murinemacrophages isolated from the peritoneal exudates, which abundantlyexpress the receptor on their surface. In addition, the reporter geneswere transferred successfully into macrophages present in the liver andspleen of intact rats using the mannose-terminal neoglycoproteincarrier.

Methods

Materials: DNA-modifying enzymes, nucleotides, and5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside were purchased fromBoehringer Mannheim (Indianapolis, Ind., U.S.A.). All chemicals,including poly(L-lysine), a-D-mannopyranosylphenyl isothiocyanatealbumin, and a-D-galactopyranosylphenyl isothiocyanate, were obtainedfrom the Sigma Chemical Company (St. Louis, Mo., U.S.A.). Luciferaseassay system was obtained from Promega (Madison, Wis., U.S.A.). Therabbit anti-β-galactosidase antibody and fluoresceinisothiocyanate-conjugated goat anti-rabbit IgG was obtained from the 5Prime to 3 Prime, Inc. All media, sera, and antibiotics were obtainedfrom Gibco Laboratories (Grand Island, N.Y., U.S.A.).

Preparation Of Mannose-Terminal Glycoprotein Carrier: Syntheticglycoprotein carriers were constructed in which poly(L-lysine), averagechain length 100 (M_(r) 20,000 Da), was glycosylated usinga-D-mannopyranosyl phenylisothiocyanate dissolved inN,N-dimethylformamide. The solution was adjusted to pH 9.5 by theaddition of 1 M Sodium carbonate, pH 9.5. Shielded from light andincubated for 16 hours at 22° C., the solution was dialyzed against 5 mMSodium chloride for two days. Approximately 0.8 to 1.0% of the amineside chains in the polylysine are glycosylated, as determined byabsorbance spectroscopy at 250 nm. As a control, an alternativeglycoprotein carrier was synthesized by substituting a-D-mannopyranosylphenylisothiocyanate with a-D-galactopyranosyl phenylisothiocyanate.

Reporter Genes And Plasmid Preparation: The expression plasmid pGEMluccontained the SV40 viral promoter and enhancer elements ligated to theP. pyralis luciferase gene. The plasmids pCMVZ and pCMVIL2r, consistingof the cytomegalovirus (CMV) promoter linked to the E. coli lacZ and theinterleukin 2 receptor genes, respectively, were also used as reportergenes. The plasmids were grown in E. coli DH5a, extracted, and purifiedby standard techniques (14). Digestions of the plasmids with restrictionendonucleases yielded the appropriate size fragments, and purity wasestablished by 1.0% agarose gel electrophoresis. The sizes of plasmidsare as follows: pGEMluc, 6.0; pCMVlacZ, 10.9; and pCMVIL2r, 5.4 kB. Nobacterial genomic DNA was present in the plasmid preparations.

Preparation Of Mannose-Terminal Glycoprotein Carrier-DNA Complexes:Complexes were formed analogously to Example 1, however, the DNA wasabout 80% supercoiled and 20% open circular.

Cells And Cell Culture: Primary macrophages were isolated from theperitoneal cavity of mice four days after the intraperitoneal injectionof one milliliter of Brewer's thioglycolate medium. The macrophages fromthe peritoneal exudate were collected as previously described, andmaintained in RPMI Media 1640. This method yielded approximately 5×10⁶cells per mouse, of which 40-75% were mononuclear phagocytes based onmorphological characteristics of the cells and cytochemicalidentification. Transfections were performed one or two days aftercollection. The isolated cells were approximately 30-60% confluent atthe time of transfection. Viability of cells was determined by serialcell counts and trypan blue exclusion.

DNA Delivery To Macrophages In Culture: One day after isolation, thecells isolated from the peritoneal exudates of mice were washed oncewith PBS (pH 7.4) and the media was changed immediately beforetransfection. The conjugate-DNA complex, containing 5 μg (0.4-0.7 pmol)plasmid, was applied to the culture medium and permitted to remain onthe cells for 24 hours unless the experiment dictated otherwise. Thecells were then either harvested for protein extraction or fixed for insitu β-galactosidase assays at several timepoints after transfection.

Animals: Adult, male Sprague-Dawley rats, weighing approximately 250 g.,were anesthetized with ether. Using aseptic technique, 0.3 to 0.6 ml ofa solution containing 300 μg (20.8-42.0 pmol) of an expression plasmidcomplexed to the carrier was injected into the caudal vena cava. Therats were killed at different intervals after infusion of the complexesand the livers, lungs, and spleens of transfected animals were removedfor analysis. Furthermore, macrophages were isolated from the alveoli,the bone marrow, and spleen. Bone marrow cells were obtained from therat's femur. The femur was surgically removed after the experimentalanimal was sacrificed, and one milliliter of media was infused into andaspirated from the marrow cavity. A single-cell suspension of the marrowwas prepared by gently aspirating the cells with a Pasteur pipette. Thecells extracted from the bone marrow were maintained in RPMI Media 1640for 8-12 hours and permitted to attach to glass slides, at which timethe adherent cells were fixed for immunocytochemical staining.Non-transfected and mock transfected animals were used as controls inall analyses. The animal research protocol was reviewed and approved bythe Case Western Reserve University Institutional Animal Care Committee.

Cytochemical Assay For β-Galactosidase Activity: Individual cellsexpressing β-galactosidase were identified following incubation with5-Bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal) as describedpreviously. Briefly, the cells were fixed with a solution of 1%glutaraldehyde in PBS for 15 minutes, and then incubated with a solutioncontaining 0.5% X-gal for 12 hours at 37° C. The cells were also stainedfor nonspecific esterase activity, which produces an insolublegrey-black dye. A minimum of 100 cells in tissue culture were counted todetermine the percentage of cells expressing β-galactosidase.

Individual cells expressing β-galactosidase in tissues were identifiedfollowing incubation with X-gal as described previously. Briefly, thecells were fixed with a solution of 0.5% glutaraldehyde in PBS for 10minutes, washed twice with PBS, pH 7.5, and then incubated with asolution containing 0.5% X-gal, 5 mM Potassium ferricyanate, 5 mMPotassium ferrocyanate, and 1 mM Magnesium chloride inphosphate-buffered saline (pH 7.4) for 6 hours at 37° C. The stainedtissues were fixed in 2% paraformaldehyde/0.5% glutaraldehyde in PBSovernight at 4° C., paraffin embedded by standard procedure, and cutinto 5 μm sections. The sections were counterstained with 0.1% nuclearfast red. The adjacent tissue sections were also stained for nonspecificesterase activity, which appears brown-black. Blue colored cells wereidentified by light microscopy.

Cytochemical Identification Of Macrophages: Cells and tissue sectionswere stained nonspecific esterase activity, which is relatively specificfor mononuclear phagocytes. The cell smears were fixed as describedabove, and incubated with a filtered solution containing a-naphthylacetate and Fast Blue BB salt for 10 minutes at room temperature. Tissuesections were stained with this solution for 1-3 hours, andcounterstained with 0.1% nuclear fast red.

Immunocytochemical Staining For β-Galactosidase: The expression of thetransgene in cells isolated from tissues (spleen and bone marrow)transfected in vivo with the plasmid pCMVZ was determined by indirectimmunofluorescence. Cell smears were fixed with methanol/acetone for 2minutes at room temperature, and the cells were incubated with a rabbitanti-β-galactosidase polyclonal antibody for one hour at 37° C. Theprimary antibody was diluted 1:100 in PBS for immunodetection in thefixed cell smears. Fluorescein isothiocyanate conjugated anti-rabbitimmunoglobulin G diluted 1:100 in PBS was used as the secondaryantibody. The cells were also counterstained with propidium iodide,which produces red fluorescence in the cell nucleus. Between eachincubation, the cells were washed three times for five minutes with PBS.The stained cells were examined by fluorescent microscopy.

Assays For Luciferase Activity: Cells in culture were harvested, lysed,and analyzed for luciferase activity as described previously. Tissueswere harvested from transfected and control rats after the animals weresacrificed and perfused in situ with 50 milliliters of cold PBS, pH 7.5.The tissues were homogenized in lysis buffer and permitted to incubateat 22° C. for 10 minutes. The cell lysates were subsequently centrifugedfor 5 minutes at 4° C., and the protein extracts were analyzed forluciferase activity. The lysates were assayed for protein content andthe measured integrated light units were standardized for total proteincontent. All measurements were performed in triplicate and expressed asan average of the values.

Statistical Analysis: Data are expressed as means±standard error of themean (SEM), and evaluated by an analysis of variance using theStudent-Newman-Keuls (SNK) test.

Results

In vitro Transfection Of Primary Macrophages Using The Mannose-TerminalGlycoprotein Carrier: Using an expression plasmid (pCMVZ) encoding theE. coli lacZ gene as a reporter gene, complexes of the plasmid and themannose-terminal glycoprotein carrier were applied to cells peritonealexudates cells isolated from mice. Twenty-four hours after transfection,the cells were examined for β-galactosidase activity. The number oftransfected cells varied from 5 to 26 per cent of all cells examined. Inaddition, the proportion of cells with non-specific esterase activity, acytochemical marker characteristic of monocytes and macrophages, thatexpressed the transgene ranged from 40% to 75%. Transfections usingcomplexes consisting of an irrelevant plasmid (pGEMluc) bound to thecarrier or the expression plasmid (pCMVZ) bound to a galactose-terminalglycoprotein carrier no significant β-galactosidase activity in theexudate cells. Faint blue staining was noted in these control cells,which was most likely due to endogenous β-galactosidase activity.Nevertheless, the percentage and intensity of blue stained cells in thecontrols was markedly less than that in the transfected dishes, Themannose-terminal glycoprotein carrier-DNA complex appeared to benon-toxic to cells since the percentage of cells viable, based on cellcounts and trypan blue staining, after treatment was not significantlydifferent than controls.

Complexes of the mannose-terminal glycoprotein carrier and theexpression plasmid pGEMluc were applied to cells isolated fromperitoneal exudates for increasing periods of time, and luciferaseactivity was measured in protein extracts of the transfected cells 24hours following transfection. As noted in the previous experiments, thelevel of expression of the transferred gene varied. An eight-foldincrease in relative luciferase activity in transfected cells waspresent (p<0.01), whereas protein extracts obtained from cells treatedwith a complexes formed using a galactose-terminal glycoprotein carrierdid not express activity significantly different than thenon-transfected control. Furthermore, the addition of a one hundred-foldmolar excess of mannosylated bovine serum albumin over complex to theculture media immediately before transfection, which should compete withthe carrier for the mannose receptor, completely inhibited the uptakeand expression of the reporter gene (p<0.01). The duration of thetransgene expression in these cells was also examined. The complexes ofthe mannose-terminal glycoprotein carrier and the expression plasmidpGEMluc were applied to cells for 24 hours, and protein extracts wereassayed for luciferase activity at several timepoints aftertransfection. Optimal transgene expression was detected one day aftertreatment, and luciferase activity decreased to control levels eightdays post transfection.

In vivo Transfection Of Macrophages Using The Mannose-TerminalGlycoprotein Carrier: The mannose-terminal glycoprotein carrier was usedto transfer reporter genes into the spleen and livers of intact animals.Rats were anesthetized, and 300 μg of plasmid (pGEMluc) was complexed tothe mannose-terminal glycoprotein carrier and infused slowly into thecaudal vena cava over several minutes. Control and mock transfections ofanimals using complexes consisting of an irrelevant plasmid (pCMVlacZ)bound to the carrier were also performed in parallel. All animalsinjected with the complex survived. Luciferase assays were performedfour days after infusion of the complexes in tissue homogenatesextracted from liver, lungs, and spleen. We observed significant levelsof transgene expression in the protein extracts from the spleen obtainedfrom transfected animals. Lower levels of luciferase activity was foundin the liver and lung. Non-transfected rats and animals treated with thecomplexes consisting of an irrelevant plasmid (pCMVlacZ) bound to themannose-terminal glycoprotein carrier had no significant luciferaseactivity in protein extracts from any tissue. Twelve days aftertransfection, luciferase activity approximated background levels in alltissues examined.

The cellular distribution of the transgene expression was examined insections of spleen and liver three days after the injection of complexescontaining pCMVlacZ. The tissues were analyzed for β-galactosidaseactivity by a cytochemical stain. An animal treated with complexes madeusing an irrelevant plasmid (pCMVIL2r) served as control.β-galactosidase expression was detected in several small cells in thespleen located in the subcapsular region, which conformed to thedistribution of cells that expressed nonspecific esterase activity basedon cytochemical staining. No β-galactosidase activity was found in thecorresponding cells of the control spleen. Rare, blue-stained cells werepresent in hepatic sections of the transfected animal, and no hepaticendothelial cells, which also have surface mannose receptors, expressedthe transgene. Nucleated cells were also isolated from the spleen andstained in vitro. Furthermore, cells extracted from the bone marrow andbronchoalveolar lavage fluid of the transfected and control animals werealso treated with a solution containing X-gal and examined forβ-galactosidase activity. Approximately 10-20 percent of the nucleatedcells obtained from the spleen stained blue. Rare cells from the mocktransfected animal were also faintly blue stained, most likely due to anendogenous β-galactosidase. Nevertheless, the percentage and intensityof blue stained cells in the controls was significantly less than thatfound in the control animal.

A polyclonal antibody directed against the bacterial β-galactosidase wasused for the immunocytochemical localization of the transgene product toestablish that the blue-stained cells in the spleen are not due toendogenous β-galactosidase or the nonspecific hydrolysis of X-gal.Nucleated cells isolated from the spleen and bone marrow of the animalsdescribed above were stained with antibody directed againstβ-galactosidase and fluorescein isothiocyanate conjugated anti-rabbitand examined for immunofluorescence. A number of the isolated cells,which were morphologically similar to the blue stained cellsdemonstrated in the cytochemical assay, had immunofluorescent staining.In addition, these cells had nonspecific esterase activity.

Discussion

We have developed a synthetic glycoprotein complex capable of mediatingtransfer of functional genes into macrophages in culture and the liversof whole animals. Expression plasmids non-covalently bound to anmannose-terminal glycoprotein carrier can be introduced efficiently intocells that express the mannose receptor. The delivery of DNA by areceptor-mediated gene transfer system is dependent on the presence ofreceptors on the surface of the targeted cell. Cells that fail toexpress the asialoglycoprotein receptor were not transfected by thissystem. In addition to macrophages, other cell types present in theperitoneal exudate that fail to express the mannose receptor, i.e.,granulocytes, lymphocytes and fibroblasts, were not transfected. Theexpression of the reporter gene was localized to cells that had eithernon-specific esterase or peroxidase activity, reliable cytochemicalmarkers used for macrophage identification.

The specificity and affinity of the ligand for the specific receptor areof considerable importance for the delivery of exogenous genes.Macrophages bind mannose-terminal glycoproteins with high affinity andspecificity. The mannose-terminal glycoprotein carrier successfullyintroduced reporter genes into macrophages in culture and in intactanimals, whereas transgene expression was not detected in cellstransfected using a galactose-terminal glycoprotein carrier. Uptake doesnot appear to be due to a non-specific increase in pinocytosis orphagocytosis secondary to the presence glycoprotein in the culturemedium. The delivery and expression of the plasmid is inhibited by theaddition of mannosylated bovine serum albumin to the culture medium,which presumably competes for the binding site(s) on the mannosereceptor. Finally, the substitution of an alternative monosaccharide formannose could increase the affinity of the DNA-carrier complex, sincethe mannose receptor also recognizes glycoproteins with glucose, fucose,and N-acetylglucosamine residues in exposed positions. In addition, genetransfer efficiency could potentially be improved by altering thecarbohydrate residue to an oligosaccharide, i.e., oligomannose, sincemonosaccharides are poorer ligands for the receptor than are polyvalentglycoproteins.

A major factor in determining the level of expression of the genestransferred into target cells involves the survival and delivery of theexogenous DNA to the nucleus. Expression of genes introduced byreceptor-mediated mechanisms may be limited by the trapping anddegradation of the complex in endosomal compartments. Mannose-terminalglycoproteins are introduced into macrophages by receptor-mediatedendocytosis, delivered to a pre-lysosomal acidic compartment, andsubsequently trafficked to the secondary lysosomes. Apparently, aportion of the introduced conjugate avoids destruction since thetransferred DNA must escape degradation after the complex has enteredthe cell in order for the transgene to be expressed. The physical stateof the DNA transferred into cells by these delivery systems may alsocontribute to its survival and subsequent expression, and highly compactform of DNA may be more resistant to nuclease digestion. Furthermore,the small size of the carrier-DNA complex may also permit theintroduction of the plasmid into the cells of the reticuloendothelialsystem specifically via the mannose receptor and not by phagocytosis.

This study illustrates the potential of specifically directing genetransfer into macrophages by targeting the mannose receptor, andtheoretically could provide an approach to the treatment of variousinborn errors of metabolism, like Gaucher disease. Pharmacologictherapies that also target the mannose receptor have been shown to beeffective in patients with Gaucher disease. Repeated treatments ofaffected individuals with modified human glucocerebrosidase, in whichthe outer carbohydrate moieties are cleaved to expose terminal mannoseresidues, have had substantial clinical improvement in their disease, asdemonstrated by reduction in hepatosplenomegaly and resolution ofanemia. Unfortunately, the cost of this therapy has been prohibitive tomany patients. Bone marrow transplantation has been shown to be curativein the non-neuropathic form of the disease, yet the potentialcomplications of transplantation precludes this procedure in manypatients, particularly those in individuals with mild disease. However,because Gaucher disease can be corrected by bone marrow transplantation,one potential approach that has been proposed for the gene therapy ofGaucher disease involves the ex vivo transfer of the normalglucocerebrosidase gene into autologous hematopoietic stem cells andtheir subsequent introduction into the patient. Alternatively,lymphoblasts could be harvested from the affected individual, infectedwith replication-incompetent, recombinant retrovirus containing thewild-type gene, and returned to the patient. The secreted enzyme wouldenter the macrophages via the mannose receptor, thus becoming thesecondary targets of therapy. In the system we describe in thismanuscript, the macrophage would be the primary target for geneticcorrection. Practical questions regarding the efficiency of genedelivery, duration and level of expression achieved using thistechnique, and the immunologic properties of the DNA-carrier complexesneed to be addressed. Nevertheless, receptor-mediated gene therapy hasthe potential of providing a non-invasive approach to the treatment ofsuch diseases.

EXAMPLE 3

We have also used a Fab fragment of an antibody directed against the ratpolymeric immunoglobulin receptor that is expressed in the airwayepithelia. The Fab peptide was covalently coupled to poly-L-lysine andcomplexed to an SV40-luciferase expression vector using the proceduredescribed below. Rats injected with the DNA complex had luciferaseactivity for as long as 8 days (the duration of the experiment) only intissues that expressed the receptor. These finding underline theflexibility of this system for delivering DNA to specific tissues of anadult animal.

Introduction

Several methods of gene transfer into the respiratory tract have beendeveloped that permits the introduction of functional genes into cellsin vivo. However, many of these approaches have lacked specificity andare cytotoxic. Replication deficient, recombinant adenoviruses have beenused to deliver the reporter genes to respiratory epithelial cells in avariety of animal models. However, the physiologic effects of treatmentwith adenovirus are not well understood, and recent evidence suggeststhat the first-generation adenoviral vectors administered at high viraltiters to animals produce a substantial inflammatory response in thelung. Liposomes have also been used to transfer functional genes to theairway epithelium, but this approach has generally been toxic to cellsand lack specificity.

Receptor-mediated gene transfer may provide a method for delivering DNAto specific target cells using a non-infectious, non-toxic vector. Thisform of gene transfer allows specific tissue targeting with DNA plasmidsof considerable size, allowing for delivery of not only the transgene,but also promoter and enhancer elements. In the case ofreceptor-mediated systems, the delivery of exogenous DNA is dependent onthe stability of the DNA-carrier complex, the presence and number ofspecific receptors on the surface of the targeted cell, thereceptor-ligand affinity and interaction, and efficient internalizationof the complex. Furthermore, expression of the transferred genes rely ontheir escape from the endosomal vesicles and trafficking to the targetcell's nucleus. The duration of transgene expression in whole animalsdelivered by exploiting receptor-mediated endocytosis has been generallybeen transient, returning to background levels within seventy-two hoursafter treatment. This has been the case for reporter genes introducedinto airway epithelial cells via the intratracheal route usingadenovirus-polylysine and transferrin-adenovirus-polylysine vectors.

We have demonstrated that in primary cultures of human trachealepithelial cells, targeting the polymeric immunoglobulin receptor (pIgR)permits the efficient delivery of the transgene specifically to cellsthat bear the receptor. The polymeric immunoglobulin receptor isexpressed only in mucosal epithelial cells, including airway epithelialand submucosal gland cells, and is specifically adapted for theinternalization and nondegradative transfer of large molecules. In thisreport, we show that targeting the polymeric immunoglobulin receptor invivo results in expression of the transgene in tissues that containreceptor-bearing cells which was maximal six days after transfection.

Methodology

Materials: DNA-modifying enzymes, nucleotides, and5-Bromo-4-chloro-3-indolyl-p-β-D-galactopyranoside were purchased fromBoehringer Mannheim (Indianapolis, Ind., U.S.A.). Luciferase assaysystem was obtained from Promega (Madison, Wis., U.S.A.). Protein A MAPSagarose columns were purchased from BioRad (Richmond, Calif., U.S.A.).Papain and poly(L-lysine) were obtained from Sigma Chemical Company (St.Louis, Mo., U.S.A.), and N-Succinimidyl-3-(2-pyridyldithio)proprionatewas from Pierce Chemical Company (Rockford, Ill., U.S.A.). The mousemonoclonal anti-human interleukin 2 receptor antibody was obtained fromDako Corporation. (Carpenteria, Calif., U.S.A.), and the fluoresceinisothiocyanate-labelled secondary goat anti-mouse antibody was fromSigma Immunochemicals (St. Louis, Mo., U.S.A.). The Vectastain ABCmethod, used in the immunoperoxidase staining procedure, was purchasedfrom Vector Laboratories (Burlingame, Calif., U.S.A.). All media, sera,and antibiotics were obtained from Gibco Laboratories (Grand Island,N.Y., U.S.A.).

Preparation Of Fab Fragments: The isolation and papain digestion ofantibodies derived from rabbits immunized with rat secretory componenthas been described previously. Briefly, polyclonal antibody was isolatedfrom rabbit serum using a Protein A MAPS agarose column as described bythe manufacturer. Isolated immunoglobulin G (2 mg) was treated with 20μg papain for 12 hours at 37° C. in the presence of 100 mM sodiumacetate (pH 5.5) 50 mM cysteine, and 1 mM EDTA. The Fab fragment wasseparated from intact antibody and Fc fragments by Protein Achromatography. An irrelevant Fab (IFab) was generated by papaindigestion of IgG from pre-immune rabbit serum.

Preparation Of Fab-Polylysine Conjugates: The Fab fragment of theanti-pIgR immunoglobulin G was covalently linked to poly(L-lysine)(M_(r) 10,000 Da) using the heterobifunctional crosslinking reagentN-Succinimidyl 3-(2-pyridyldithio)proprionate (SPDP). The Fab fragmentwas incubated with a seventy-five fold molar excess of SPDP in 0.1Mphosphate buffered saline (PBS), pH 7.5, at 22° C. for 60 minutes. Afterintroduction of 2-pyridyl disulfide structures onto the Fab fragment,unreacted SPDP and low molecular weight reaction products were removedby dialysis. The disulfide bridges of the modified Fab fragment werecleaved with 25 mM dithiothreitol. Both the poly(L-lysine) and SPDP wasadded in fifteen fold molar excess to the modified Fab fragment, and thereaction was carried out at 22° C. for 24 hours. The conjugate wasdialyzed to remove low molecular weight reaction products, and analyzedby separating the resultant proteins on a 0.1% SDS-7.5% polyacrylamidegel electrophoresis. As described previously, analysis of the conjugatedemonstrated a protein that migrated slowly, corresponding to a proteingreater than 200 kDa in size.

Reporter Genes And Plasmid Preparation: The expression plasmid pGEMluccontained the SV40 viral promoter ligated to the P. pyralis luciferasegene. The plasmids pCMVZ and pCMVIL2r, consisting of the cytomegalovirus(CMV) promoter linked to the E. coli lacZ and the interleukin 2 receptorgenes, respectively, were also used as reporter genes. For studies ofluciferase activity, these plasmids were employed as irrelevant DNA(IDNA) controls. The plasmids were grown in E. coli DH5a, extracted, andpurified by standard techniques. Digestions of the plasmids withrestriction endonucleases yielded the appropriate size fragments, andpurity was established by 1.0% agarose gel electrophoresis. The sizes ofplasmids are as follows: pGEMluce, 6.0; pCMVlacZ, 10.9; and pCMVIL2r,5.4 kB. No contamination with bacterial genomic DNA or RNA was presentin the plasmid preparations.

Preparation Of Fab-Polylysine-DNA Complexes: The carrier-DNA complexeswere formed using a method described previously.

Animals: The anti-rat secretory component Fab antibody-polylysinecarrier was used to transfer reporter genes into the airways and liversof intact animals. Adult, male Sprague-Dawley rats, weighingapproximately 250 g., were anesthetized. Using aseptic technique, 0.3 to0.6 ml of a solution containing 300 μg of an expression plasmidcomplexed to the carrier was injected into the caudal vena cava. Therats were sacrificed at several different times after infusion of thecomplexes and various organs were removed for analysis. Mocktransfections of animals using complexes consisting of an irrelevantplasmid bound to the carrier or the expression plasmid bound to acarrier made with an irrelevant Fab fragment were also performed inparallel. The animal research protocol was reviewed and approved by theCase Western Reserve University Institutional Animal Care Committee.

Cytochemical Assay For β-Galactosidase Activity: Individual cellsexpressing β-galactosidase in tissues were identified followingincubation with 5-Bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal)as described previously. Briefly, the cells were fixed with a solutionof 0.5% glutaraldehyde in PBS for 10 minutes, washed twice with PBS, pH7.5, and then incubated with a solution containing 0.5% X-gal, 5 mMPotassium ferricyanate, 5 mM Potassium ferrocyanate, and 1 mM Magnesiumchloride in phosphate-buffered saline (pH 7.4) for 4 hours at 37° C. Thestained tissues were fixed in 2% paraformaldehyde/0.5% glutaraldehyde inPBS overnight at 4° C., paraffin embedded by standard procedure, and cutinto 5 μm sections. The sections were counterstained with nuclear fastred. Blue colored cells were identified by light microscopy. A minimumof 100 cells were counted to determine the percentage of cells persection that express β-galactosidase. In addition, adjacent sectionswere stained with Alcian blue/periodic acid Schiff or haematoxylon/eosinusing standard protocols.

Assays For Luciferase Activity: Cells in culture were harvested, lysed,and analyzed for luciferase activity as described previously. Tissueswere harvested from transfected and control rats after the animals weresacrificed and perfused in situ with cold PBS, pH 7.5, for five minutes.The tissues were homogenized in lysis buffer and permitted to incubateat 22° C. for 10 minutes. The cell lysates were subsequently centrifugedfor 5 minutes at 4° C., and the protein extracts were analyzed forluciferase activity. The lysates were assayed for protein content andthe measured integrated light units (10 second interval) werestandardized for total protein content. All measurements were performedin triplicate and expressed as an average of the values.

Immunohistochemical Staining For The Interleukin 2 Receptor: Theexpression of the transgene in tissues transfected with the plasmidpCMVZ was determined by indirect immunofluorescence. Frozen sections ofvarious tissues were fixed with acetone for 10 minutes at -20° C., andtreated with for ten minutes at 22° C. to reduce autofluorescence. Thesections were then incubated with 10% goat serum in PBS, pH 7.5, for onehour at room temperature. The cells were incubated sequentially with amouse monoclonal anti-interleukin 2 receptor antibody and fluoresceinisothiocyanate-conjugated goat anti-mouse IgG. Both antibodies werediluted 1:100 in PBS, and between each incubation, the cells were washedthree times for five minutes with PBS, pH 7.5. The stained cells wereexamined by fluorescent microscopy.

Results

In vivo Transfection Using The Anti-Secretory Component FabAntibody-Polylysine Carrier: All animals injected with the anti-ratsecretory component Fab antibody-polylysine carrier-DNA complexsurvived. Luciferase assays were performed 48 hours after infusion ofthe complexes in tissue homogenates extracted from liver, lungs, spleen,and heart. We observed significant levels of transgene expression in theprotein extracts from the liver and lungs obtained from transfectedanimals. No detectable luciferase activity was found in the spleen andheart, tissues that do not express the pIgR. Furthermore, animalstreated with the complexes consisting of an irrelevant plasmid(pCMVlacZ) bound to the carrier or the expression plasmid (pGEMluc)bound to a carrier based on an irrelevant Fab fragment resulted in nosignificant luciferase activity in any tissue examined. Thus, onlytissues that contain cells bearing pIgR are transfected, andtransfection cannot be attributed to the nonspecific uptake of anirrelevant Fab antibody-based complex.

A time course of the expression of the transferred gene, in whichluciferase activity in protein extracts derived from the four tissueswas measured at different timepoints after injection of the complex, wasdeveloped. Luciferase activity persisted in the liver and lung, tissueswhich have pIgR, achieving maximum values of 13795±4431 and461402±230078 integrated light units (ILU) per milligram of proteinextract, respectively, at four to six days after injection. Tissues thatfailed to express the receptor did not have significant transgeneexpression.

The cellular distribution of the transgene expression was examined insections of various tissues. Three days after the injection of complexescontaining pCMVlacZ, tissue sections of trachea, lung, and liverunderwent cytochemical staining for β-galactosidase activity. An animaltreated with complexes made using an irrelevant plasmid (pCMVIL2r)served as control. Expression in the trachea was limited to the cellslining the epithelial surface. No β-galactosidase activity was detectedin the tracheal sections from the mock transfected animal. Theexpression of the transgene was variable, and in some areas of therespiratory epithelium greater than 50% of the cells stained blue. Ingeneral, expression ranged from 10-20% of the tracheal epithelial cells.Both ciliated and secretory (goblet) respiratory epithelial cellsexpressed β-galactosidase activity, based on Alcian blue/periodic acidSchiff staining of adjacent sections of the airway. No expression fromthe transgene was detected in the terminal airways or alveoli in eitherthe transfected or control animal (data not shown). This conforms to thedistribution of epithelial cells that express the pIgR based on in situimmunohistochemical staining. Rare submucosal glands were evident in thetracheal sections, and only faint blue staining was noted. Noinflammatory response was found in any of the tracheal sections from thenon-, mock-, and transfected animals. In addition, a mouse monoclonalantibody directed against the human interleukin 2 receptor, a surfaceprotein that has been used as a reporter in the transduction ofrespiratory epithelial cells in vitro but is not naturally expressed inthese cells, was used for immunofluorescent localization of thetransgene product in the trachea of the animal transfected with theplasmid pCMVIL2r. Serial sections of the trachea were examined for thepresence of fluorescence, and the apical membrane of numerousrespiratory epithelial cells from the transfected animal stainedappropriately. No specific fluorescent staining was detected in theairway epithelia of an animal mock-transfected with pCMVlacZ. Rare,blue-stained hepatocytes were also found in hepatic sections of thetransfected animal. Transgene expression was not identified in thelivers from either non- or mock-transfected rats.

Discussion

We report the successful transfer of reporter genes into the airwayepithelium in vivo following the injection of a targeting complexconsisting of the Fab portion of immunoglobulin G directed against therat polymeric immunoglobulin receptor conjugated to poly(L-lysine), andnoncovalently bound to plasmid DNA. This technique specificallydelivered the transgene to the liver and lung, tissues in which thisreceptor is expressed. Other tissues that do not express the receptor,like the spleen and heart, were not transfected. In addition, followinginjection of a conjugate prepared with irrelevant Fab fragments noexpression was detected, and a complex prepared with a plasmidcontaining an irrelevant reporter gene also failed to produce detectableluciferase activity. Thus, this complex specifically targetsreceptor-bearing tissues and the normal trafficking of the receptor'snatural ligands does not interfere with the uptake of the transgene invivo.

Most of the strategies for gene transfer into the respiratory tractcurrently available depend on viral vectors which do not specificallytarget respiratory epithelial cells, and rely upon the intratrachealroute of delivery to permit targeting of the airway. Intratrachealinstillation has also been used to specifically direct gene transfer byother means, like liposomes and adenovirus-transferrin-polylysineconjugates, to the airway epithelium. Systemic delivery of DNA bound tocationic liposomes has not been selective and transfers functional genesto a number of cell types in different tissues. The specificity ofreceptor-mediated gene transfer for cells that bear the pIgR may beuseful in targeting defective cells in the airways of patients withcystic fibrosis.

EXAMPLE 4

Familial hypercholesterolemia (FH) is a human genetic diseasecharacterized by fulminant atherosclerosis and cardiovascular disease. Amutation in the gene for the receptor that mediates the uptake of thelow density lipoprotein (LDL) is responsible for this disease. One inevery 500 people is heterozygote for a mutation in the LDL receptor genethat is responsible for FH. As a result, LDL is removed from theirplasma at only two thirds the normal rate. In the fourth to fifth decadeof life, the elevated levels of LDL in plasma cause symptomaticatherosclerosis in these patients. FH-homozygotes (one in a millionpeople) have little or no functional LDL receptor, depending on thedomain of the protein that is affected by the mutation. This results insymptomatic coronary atherosclerosis before the age of 20. Treatmentwith bile acid-binding resins and inhibitors of cholesterol synthesishas been considerably successful in heterozygous FH patients bystimulating the production of LDL receptor from the single normal gene.In FH homozygotes there is no response to drug therapy. Because of theabsence of a normal gene that can be stimulated, the replacement of themutated gene is the only possible approach for the treatment ofhomozygous FH patients. Since the liver is the major organ responsiblefor LDL catabolism, the two approaches taken for the treatment of thedisease target this organ: liver transplantation and gene therapy.Transplantation of a normal liver into a patient with FH can correcthyperlipidemia, suggesting that reconstitution of the hepatic LDLreceptor should be sufficient for phenotypic improvement. Based on thisresults, all the approaches undertaken using gene therapy for thetreatment of FH have targeted the hepatocytes.

In order to understand the mechanism of disease, it is necessary to beaware of the metabolism/fate of cholesterol in the organism. Every cellneeds cholesterol for the synthesis of the plasma membrane. The adrenalglands and the corpus luteum in the ovary, in addition, requirecholesterol for the synthesis of steroid hormones. The liver is theorgan with the highest demand because of the production of bile acids.Cholesterol is obtained in peripheral tissues either fromreceptor-mediated uptake of low density lipoproteins (LDL), which arethe main carriers of endogenous cholesterol in the blood, or bybiosynthesis. HMG CoA reductase is the rate-determining enzyme in thepathway. Dietary cholesterol is carried in the bloodstream bychylomicron particles, which are taken up by specific receptors in theliver. In order to provide the different tissues with cholesterol, theliver secretes very low density lipoprotein (VLDL) particles composed oftriglycerides, cholesteryl esters and apoproteins C, E and B-100. Theuptake of triglycerides from VLDL by adipose tissue and muscle convertsthese particles into intermediate density lipoproteins (IDL). The LDLreceptor, present at highest concentration in the liver and adrenalglands but also in the rest of tissues, recognizes the apo E and apoB-100 components of IDL. Thus, under normal conditions IDL is mostlycleared from the bloodstream by LDL receptor-mediated uptake. Theremaining IDL is converted to LDL, which is taken up as well by the LDLreceptor that recognizes the apo B-100 component. The clearance ofcholesterol from the organism is carried out by the liver, where it isconverted to bile acids and secreted into the digestive tract. Althoughmost of the cholesterol is reabsorbed in the terminal ileum for liverreutilization, this pathway provides the route of exit.

Thus, the presence of non-functional LDL receptors that are unable toclear IDL and LDL from the blood results in elevated serum LDL levels,and therefore total serum cholesterol. This is responsible forcholesterol deposition in the artery walls and thus, atherosclerosis.

The Watanabe Heritable Hyperlipidemic (WHHL) rabbit has been previouslyused to study the effectiveness of gene therapy techniques in correctinghypercholesterolemia. A 12 nucleotide in-frame deletion in theligand-binding domain of the LDL receptor, similar to one class ofmutation found in FH patients, results in symptoms, evolution andhistopathology that parallel those of FH.

Materials and Methods

Construction of the DNA Plasmids

The plasmid DNAs used in this work are pLDLR-17, PCK-hLDLR, PCK-rLDLRand SV40-luciferase. pLDLR-17 was provided by Dr. David Russell(University of Texas, Medical Center, Dallas) and consists of thecytomegalovirus (CMV) promoter/enhancer linked to the human LDL receptorcDNA. It contains a fragment of DNA corresponding to the 5' untranslatedregion (UTR) of the Alfalfa Mosaic Virus 4 (AMV4) RNA linked to thehuman LDL receptor cDNA. This sequence acts as a translational enhancerby decreasing the requirements for initiation factors in proteinsynthesis. The PCK-hLDLR plasmid has been constructed by subcloning thehLDL receptor cDNA from the pLDLR-17 into a pTZ18R vector (Pharmacia)containing the phosphoenolpyruvate carboxykinase (PEPCK) promoter (-460to +73) and an intron and polyadenylation signal from the simian virus40 (SV40) small T antigen. In a two step process, the hLDL receptor cDNAwas excised with SacI and SmaI from the pLDLR-17 and blunted using T4DNA polymerase. The blunted fragment was subcloned into the HincII siteof a pTZ18R vector. The cDNA was then excised with XbaI and SalI andintroduced into the homologous sites of the pTZ18R-PEPCK promoter-SV40polyA plasmid. For the construction of pPCK-rLDLR, the EcoRI-EcoRIfragment from prLDLR-9 (provided by Dr. James Wilson, University ofPennsylvania) containing the rabbit LDL receptor cDNA was subcloned intothe EcoRI site of a pBluescript (Stratagene). This construct wasdigested with SacI and blunted and then digested with XbaI, anddirectionally subcloned into the XbaI-blunted HindIII sites of a pTZ18Rvector containing the PEPCK promoter (-460 to +73) and an intron andpolyadenylation signal from SV40 small T antigen. The SV40-luciferaseplasmid (Promega) contains the SV40 viral promoter and enhancer ligatedto the P. pyralis luciferase gene inserted into the pUC19 vector(Pharmacia).

Formation of the Poly-L-Lysine-DNA Complex

Production Of The Galactosylated poly-L-Lysine: Poly-L-lysine wasgalactosylated as described (PNAS). Two mg of poly-L-lysine-HBr (SigmaP-7890, average chain length, 100) was reacted with 85 mg ofa-D-galactopyranosyl phenyl-isothiocyanate (Sigma G-3266). The solutionwas adjusted to pH 9 by the addition of 1/10 volume of 1 M sodiumcarbonate pH 9. The tube was shielded from light by aluminum foil andmixed for 16 hours at room temperature, then dialyzed using Spectra-Pordialysis tubing (3500 M.W. cutoff) against 500 ml of 5 mM NaCl for 2days with frequent changes of buffer (4 changes/day). The reaction isstoichiometric and resulted in the galactosylation of 0.8 to 1% of theNH₃ groups present in the solution.

Basic Protocol For The Condensation Of DNA: Plasmid DNA was preparedusing standard techniques. The DNA was resuspended in 10 mM Tris-HCl, pH8.0, containing 1 mM EDTA and the concentration of the DNA determinedspectrophotometrically. The DNA preparation was treated twice with RNAseA+T1. This step ensures that RNA is not present in the solution (RNAinhibits the condensation of DNA by poly-L-lysine). A solutioncontaining a high concentration of DNA (1.5-2 mg/ml) was used in furthersteps. An example of a typical protocol for DNA condensation isdescribed as follows:

a) 300 mg of DNA in 200 ml of 0.75 M NaCl (added from 5 M NaCl solution)is vortexed at medium speed, using a VIBRAX apparatus (IKA-VIBRAX-VXR).This step is necessary to increase the effective length of the DNApolymer in high salt solutions, thus achieving efficient binding of thepoly-L-lysine moiety to the DNA backbone.

b) 120 mg of poly-L-lysine or galactosylated poly-L-lysine (averagechain length 100) in 200 ml of 0.75 M NaCl (added from a 5 M NaClsolution) is added dropwise over a period of 30 minutes to 1 hour in 5μl aliquots. This amount translates into a molar ratio of 1 DNA PO₄ ⁻group to 1 carrier NH₃ ⁺ group.

c) The solution becomes turbid at the end of the process. Three μlaliquots of 5 M NaCl are added dropwise to the vortexing solution untilturbidity disappears as monitored by eye. This process is slow, allowing60 seconds between the addition of each new aliquot of 5 M NaCl. Thenthe solution is subjected to circular dichroism (CD) spectroscopicmonitoring. The solutions of DNA/poly-L-lysine complexes were alsoanalyzed using a JEOL-100C electron microscope. The condensation processis complete when the diagnostic spectrum of the DNA complex is observedand is further established by EM. For subsequent preparations of DNAcomplex consisting in the same plasmid DNA at the same concentration ofnucleotide, the protocol can be followed without monitoring with CD.When using different concentration of DNA or a different plasmid the CDmonitoring should be repeated.

Animals

Six adult male Watanabe rabbits (2.8-3.2 Kg of bodyweight) were used inthese studies. These animals have been purchased from an establishedcolony at the National Institutes of Health. In order to introduce theDNA complex into the animal, we perform a single injection of 3-10 ml ofthe DNA-complex solution (˜400-900 mM NaCl) into the marginal ear veinof the rabbit. Approximately 1.5 ml of blood was drawn from the earartery at 4 p.m. The determination of the concentration of serumcholesterol was performed in the Clinical Laboratory of UniversityHospitals of Cleveland from 300 μl of serum. At different time pointsfollowing the introduction of the DNA complex, a rabbit was subjected toa liver biopsy. Total DNA was isolated from the hepatic sample andsubjected to PCR amplification in order to detect the presence of thetransferred DNA. Rabbit #774 was treated with lovastatin (Mevacor, Merckand Dohme) orally at a dose of 10 mg per day.

Polymerase Chain Reaction (PCR) Amplification

In order to detect the presence of the transferred DNA in the liver ofthe treated animal, total DNA was isolated from the hepatic sampleobtained upon biopsy. In the case of rabbit #737, the DNA of interestwas then amplified by PCR using an upstream primer corresponding topositions 32-50 in exon 1 of the 5' UTR of the PEPCK gene and adownstream primer complementary to nucleotides 589-607 of the human LDLreceptor cDNA. The amplified fragment corresponds to a 1100 bp band uponhybridization with a 700 bp fragment corresponding to the 5' end of thehuman LDL receptor cDNA labeled with 32P-dCTP. Appropriate primerscorresponding to the chimeric CMV-hLDL receptor gene will be used forthe PCR amplification of the transferred plasmid from liver tissueobtained from rabbit #774.

Elisa

Aliquots of 75 μl corresponding to 1 μg of DNA of either newly preparedgalactosylated-poly-L-lysine/DNA complex, plasmid DNA orgalactosylated-poly-L-lysine were incubated overnight at 4° C. to coateach well of a 96 well microtiter plate. The next day the wells werewashed 3 times with phosphate-buffered saline (PBS), then blocked for 2hours at 37° C. with 5% bovine serum albumin (BSA) in PBS and washed 3times with the washing buffer containing 1% BSA and 0.5% Tween-20 inPBS. Seventy-five μl of serum from rabbit #774 obtained at differenttime points before and after the repeated administration of the DNAcomplex at dilutions of 1:3 and 1:30 were added to the wells andincubated for 90 minutes at 37° C. The wells were then washed withwashing buffer and incubated with the secondary antibody at 1:3000dilution. The secondary antibody consists of a mouse monoclonal antibodyagainst rabbit immunoglobulins conjugated to alkaline phosphatase(Sigma). After a final wash with washing buffer, the pNPP substrate at 1mg/ml in glycine buffer was added to the wells to develop the reactionand spectrophotometric readings at 410 nm were taken in a Dynatechautomated ELISA reader. Values taken at 120 minutes were chosen forcomparison.

Results

1. Rabbit #676: injection of the poly-L-lysine/DNA complex containing 3mg of the chimeric PCK-hLDLR gene.

In a first set of experiments, we condensed 3 mg and 9 mg of pPCK-hLDLRwith galactosylated poly-L-lysine using the techinque developed in ourlaboratory and we injected them into the peripheral circulation ofWatanabe rabbits.

The promoter from the gene for the cytosolic form of thephosphoenolpyruvate carboxykinase (PEPCK) from the rat has beencharacterized in detail. This promoter was used in these experimentsbecause it is expressed at a high level in the liver and its expressioncan be controlled by diet and hormones. Starvation and a high protein,carbohydrate-free diet stimulate PEPCK gene transcription while a highcarbohydrate diet reduces transcription from the PEPCK promoter. Inaddition, cAMP and glucocorticoids induce, and insulin inhibits,expression of the PEPCK gene in the liver. The PEPCK promoter is thussuitable for the regulation of a linked structural gene introduced intothe liver and was used in our first experiments for the hepaticexpression of LDL receptor.

In our first approach we have injected the poly-L-lysine/DNA complexcontaining 3 mg of DNA. This basic dose of DNA was decided based onprevious experiments performed in rats. As shown in FIG. 13, theadministration of a DNA complex solution containing 3 mg of thepPCK-hLDLR plasmid in a relaxed state to rabbit #676 did not result in asignificant decrease in total serum cholesterol levels. A secondinjection of DNA complexes appropriately condensed containing 3 mg ofthe same DNA caused a 20% reduction of the levels of cholesterol in theblood. Four weeks after this second administration, cholesterol returnedto approximately pre-treatment levels, reaching a peak at about 35 days.

A 20% decrease in total serum cholesterol levels resulting from theexpression of the PCK-hLDL receptor gene will likely be helpful but willnot totally alleviate the disorder in FH patients. The number ofpoly-L-lysine/DNA complexes corresponding to 3 mg of DNA that we haveintroduced into the animal in our first approximation to theseexperiments accounts for 0.01% of the total number of asialoglycoproteinreceptors in the liver. Consequently, a linear correlation betweenincreasing concentration of DNA complexes and expression of the PCK-hLDLreceptor gene is to be expected.

2. Rabbit #737: injection of the poly-L-lysine/DNA complex containing 9mg of the chimeric PCK-hLDLR gene.

In our second experiment, 9 mg of the PCK-hLDLR gene appropriatelycondensed with galactosylated poly-L-lysine were administered to rabbit#737. As shown in FIG. 14, the treatment resulted in a 38% reduction oftotal serum cholesterol levels which lasted for about 5 weeks. Thus, a3-fold increase in the dose of DNA complex resulted in a 2-foldreduction in total serum cholesterol levels.

3. Rabbit #16: injection of the DNA complex containing 3 mg of theCMV-hLDLR gene.

The promoter for the cytosolic form of the PEPCK gene has the advantageof driving expression in the liver almost specifically and in aregulated fashion. Although they are neither physiologic nor regulated,viral promoters confer high levels of expression to linked structuralgenes. The chimeric CMV promoter/enhancer has been used with success forgene therapy in WHHL rabbits using adenoviruses for gene transfer.Recently, Kozarsky et al. have reported that the CMV promoter/enhancerand the chimeric β-actin/CMV promoter were the promoters of choice inorder to obtain highest expression of the human LDL receptor genetransferred to WHHL rabbits using adenoviral infection. Based on theseobservations, we injected the chimeric CMV-hLDLR gene in order toincrease the level of expression of the human LDL receptor gene in theliver of WHHL rabbits.

The administration of a DNA complex solution containing 3 mg of thechimeric CMV-hLDL receptor gene to rabbit #16 resulted in a maximalreduction of 30% in total serum cholesterol levels (FIG. 15). Elevenweeks after the injection cholesterol levels are still 20% below thoseobserved before the treatment.

4. Rabbit #775: repeated administration of the DNA complex containing 3mg of pCMV-hLDLR.

Three mg of pCMV-hLDLR contained in a DNA complex solution were injectedinto rabbit #775, causing a maximal 24% reduction in the concentrationof cholesterol in the blood 3 weeks after the treatment (FIG. 16A).

The life-span of hepatocytes is reported to be about 108-150 days, sothat the persistence of the introduced DNA is limited. Furthermore, alarger therapeutic effect may be of interest after a single injection ofthe DNA complex. Thus, it may be necessary to inject a patient multipletimes to ensure the appropriate level of LDL receptor in the liver. Wetested the effect of injecting the DNA complex several times into thesame animal. Rabbit #775 has been reinjected twice with 3 mg of thepCMV-hLDLR DNA complex being each injection spaced by 3 weeks. Therepeated administration of the complex did not result in a furthersignificant reduction in total serum cholesterol levels.

5. Rabbit #774: repeated administration of the DNA complex containing 3mg of pCMV-hLDLR.

Rabbit #774 was injected with 3 mg of the pCMV-hLDLR complex. Weobserved a 36% decrease in the cholesterol levels in the blood (FIG.16B). To date four reinjections once every 2 weeks have been performedwith the same amount of DNA complex. Two of them have resulted in aminimal effect while the other two in a null reduction of total serumcholesterol levels. However, after five administrations of the DNAcomplex solution containing 3 mg of pCMV-hLDLR, the concentration ofcholesterol has dropped about 48% with respect to pre-treatment levels.

6. Administration of lovastatin to rabbit #774: inhibition of theendogenous synthesis of cholesterol.

As described in the introduction, there is a pathway for cholesterolsynthesis inside the cell. A failure in repressing this metabolicpathway even when the hepatocyte is supplied with cholesterol throughthe uptake by the human LDL receptor could possibly inhibit furtherclearance of cholesterol. Lovastatin is a known inhibitor of HMG CoAreductase, the rate-limiting enzyme in the synthesis of cholesterol.Thus, the treatment with this drug of a rabbit that has been injectedrepeated times with the DNA complex should indicate if cholesterolsynthesis was the limiting factor for a further reduction of total serumcholesterol levels. Rabbit #774 has been treated with 10 mg oflovastatin per day for 10 weeks. A futher 20% reduction in the levels ofcholesterol has been observed. The inhibition of the endogenous pathwayfor cholesterol synthesis has thus brought the cholesterol concentrationof rabbit #774 to 40% of that prior the first gene transfer (FIG. 16B).

7. Injection of the DNA complex containing an irrelevant DNA.

In order to control for a possible artifactual reduction in total serumcholesterol levels by injecting rabbits with the galactosylatedpoly-L-lysine/DNA complexes in a solution with high NaCl concentration(˜900 mM), we have administered a DNA complex solution containing anirrelevant DNA such as the luciferase gene into rabbit #775. FIG. 17shows that the injection results in a non-significant (≧12%) andtransient (≧5 days) reduction in the serum cholesterol concentration. Inaddition, we have also injected inappropriately condensed DNA complexesencoding the PCK-hLDLR gene. They result in a null or minimal andtransient decrease in the cholesterol levels in the blood as well. Thus,we have confirmed that the reduction in total serum cholesterol levelsafter the injection of appropriately condensed DNA particles encodingthe human LDL receptor gene are not a result of either the high NaClconcentration of the solution or the presence of galactosylatedpoly-L-lysine/DNA particles.

8. Detection of the transferred DNA in the liver of rabbit #774.

The DNA complex used in this project is targeted to the hepaticasialoglycoprotein receptor using galactose as a ligand. It is knownthat macrophages have a similar receptor which is able to cleargalactosylated particles larger than 15 nm from the bloodstream.

In order to prove that the human LDL receptor DNA was delivered to thehepatocytes, we performed a liver biopsy in rabbit #737 60 days afterthe injection of 3 mg of the PEPCK-hLDL receptor gene. Total DNA wasisolated and subjected to PCR amplification with the primers describedabove, together with total DNA from the liver of a non-injected rabbit.The expected band of 1,100 bp was detected in the lane corresponding tothe treated rabbit but not in the non-treated animal.

9. Evaluation of the immune response of rabbit #774 after the repeatedadministration of the poly-L-lysine/DNA complex.

In the field of gene therapy, immunogenicity of the delivery vehicle isoften a concern. While retroviral vectors can escape detection by theimmune system, it has been reported that adenoviral vectors do not. Thesuccess of a second administration of adenoviral particles for thetransfer into Watanabe rabbits of the human LDL receptor gene wasblocked by the onset of an immune response against the viral proteins(REF Kozarsky).

The system for receptor-mediated gene transfer has not been studied indepth in regard of its immunogenicity. It has been reported that afterthe repeated administration of an asialoorosomucoid-poly-L-lysine/DNAcomplex into mice, neutralizing antibodies against the asialoorosomucoidand poly-L-lysine components of the complex but not against the DNA canbe detected at a dilution 1:1000 (REF). Ferkol et al. also reported thedetection of circulating antibodies at a 1:2000 dilution against the Fabfragment-poly-L-lysine but not the DNA moiety of a complex upon repeatedadministration into mice.

We thus needed to test if the use of galactosylated-poly-L-lysine forthe condensation of DNA was immunogenic as well. For this purpose, thepresence of antibodies against the galactosylated-poly-L-lysine-DNAcomplex was evaluated in sera obtained from rabbit #774 at differenttime points before and after the repeated administration of the complex.In a first experiment, the DNA complex solution containing 1 μg of DNAwas adsorbed to the wells of a microtiter plate and then incubated withsera at dilutions 1:3, 1:30 and 1:300. Bound antibodies were detectedwith an anti-rabbit secondary antibody conjugated with alkalinephosphatase. There is an increase of antibodies in the serum of rabbit#774 upon repeated administration of the DNA complex. In fact, theystart to be detectable after the third injection of the DNA complex butnot after the first or the second. In addition, it has to be emphasizedthat only at dilutions 1:3 and 1:30 could a response be detected.

A second experiment was performed in order to establish which moiety ofthe DNA complex is responsible for inducing the weak though clear immuneresponse. We then adsorbed to the microtiter plate wells either 1 μg ofDNA, freshly prepared DNA complex containing 1 μg of DNA or thecorresponding amount of galactosylated-poly-L-lysine. The results showthat the galactosylated-poly-L-lysine moiety accounts almost entirelyfor the induction of an immune response against the complex in Watanaberabbits.

Discussion

The data presented here strongly suggest that the method has been ableto at least partially correct hyperlipidemia in WHHL rabbits.

FIGS. 13-16 clearly show that a single injection of the DNA complexcontaining the human LDL receptor gene results in a significant decreaseof total serum cholesterol levels in WHHL rabbits. This reduction rangesfrom 20% in rabbit #676 to 38% in rabbit #737. In contrast, we show thatthe administration of a non-relevant plasmid DNA such aspSV40-luciferase (FIG. 17) or of a human LDL receptor-encoding plasmidthat is not appropriately condensed (FIG. 17) results in a null ornon-significant decrease in serum cholesterol.

We have used two different promoter regions for the regulation ofexpression of the human LDL receptor gene. It is tentatively suggestedthat the CMV regulatory region confers higher levels of expression inthe liver of rabbits than the promoter for the cytosolic form of the ratPEPCK gene. This observation may not be correct for every species. PEPCKactivity in the liver of rabbits is characterized by being only 10% dueto the cytosolic isozyme. In addition, stimulation of the cytosolic generesults in only a 2-fold induction of activity. Thus, the PEPCK promotermay not be the best choice for this species. But the use of aphysiologic and tightly regulated promoter as the one for the PEPCK genemay well be the one of choice over a strong but viral promoter as theCMV in other species or for the treatment of other genetic diseases.

In order to determine the time-course of the therapeutic effect rabbits#676, #737 and #16 were subjected to a single injection of the DNAcomplex containing the human LDL receptor gene. The reduction in thelevels of cholesterol in the blood persisted for 4 weeks in rabbit #676and for 5 weeks in rabbit #737. Based on previous experiments performedin rats where the expression of the transfected pPEPCK-human Factor IXgene was shown for up to 140 days, we were expecting a longer durationof the effect. Different factors can explain this premature terminationof the corrective effect of hyperlipidemia. It is well known thatrabbits are highly immunogenic and that rats are not. The synthesis inthe WHHL rabbits of a human protein after the introduction of the humanLDL receptor gene could possibly trigger an immune response against theforeign protein, although there is an 80% homology between both speciesat the protein level. In addition, hepatocytes seem to have a limitedlife-span. Some studies in the rat indicate that the life-span ofhepatic cells is 108-150 days. Based on this observation, 40% of theincrease in cholesterol levels 5 weeks after the introduction of the DNAcomplex could result from the physiological turnover of liver cells.However, this fact cannot account for 100% of the increase. In addition,it would contradict with the long-term expression observed in ratsinjected with pPEPCK-human FIX. Another possible explanation for thepremature termination in the therapeutic effects resulting from theexpression of the human LDL receptor gene would be inactivation ordegradation of the transferred DNA.

The theoretical number of poly-L-lysine-DNA complexes that can be formedwith 3 mg of DNA accounts for 0.01% of the total number ofasialoglycoprotien receptors in the liver. Consequently, we would expectthat an increase in the dose of DNA complex results in an enhancedtherapeutic effect. To study the dose-response relationship, we haveinjected rabbit #676 with 3 mg of pPCK-hLDLR and rabbit #737 with 9 mgof the same DNA. As shown in FIGS. 13 and 14, a 3-fold increase in thedose of DNA complex results in a 2-fold higher reduction in cholesterollevels. Although these data do not establish linear correlation, anincrease in the dose clearly results in an enhanced response.

If we consider the poly-L-lysine/DNA complex as a potential drug, it isdesirable to be able to repeatedly administer it to the same animal. Forthis reason, rabbit #774 has been subjected to repeated administrationof 3 mg of the CMV-hLDLR DNA once every 2 weeks. After an initialdecrease of 36% in serum cholesterol levels following the firstinjection, the effect of the repeated administration of the DNA complexhas not been consistent. Rabbit #775 has been treated 3 times with 3 mgof the CMV-hLDLR DNA. Again, after an initial 24% reduction in thecholesterol levels, the second and third treatments have not resulted ina clear effect. We can find three possible explanations for theseresults. First, that the DNA complexes were not appropriately condensed.DNA upon condensation with poly-L-lysine can result in three differentstructures: aggregated (condensed particles out of solution), tightlycondensed and relaxed. Only DNA tightly condensed into small particlesis effective in delivering genes in vivo. Second, that the rabbits areproducing neutralizing antibodies against the vehicle. We have somepreliminary data regarding the immune response of rabbit #774 againstthe poly-L-lysine-DNA complex. Third, further clearance of cholesterolfrom the blood is limited by an impairment in the endogenous metabolismof cholesterol in the hepatocyte of the mutant Watanabe rabbit. In orderto test this last hypothesis, rabbit #774 was treated with lovastatin(10 mg/day), a known inhibitor of HMG CoA reductase, for 10 weeks. Theobservation of a further 20% reduction in the cholesterol concentrationsuggests that the inhibition of cholesterol synthesis in the hepatocyteis not complete even when the cell is supplied with cholesterol uponuptake of LDL by the heterologous LDL receptor.

Preliminary results regarding the immunogenicity of thegalactosylated-poly-L-lysine/DNA complex indicate that the repeatedadministration triggers the onset of an immune response in the Watanaberabbit. They also show that circulating antibodies can recognize thegalactosylated-poly-L-lysine but not the DNA moiety. These results agreewith previous reports regarding the immunogenicity of anasialoorosomucoid-poly-L-lysine/DNA complex and of anFab-poly-L-lysine/DNA complex. Though it is clear that the complexdesigned in our laboratory can in fact elicit an immune response uponrepeated administration in the same animal, it has to be noticed that wecould only detect circulating antibodies at much lower dilutions (1:3and 1:30 as compared to 1:1000 and 1:2000 in their case). Thisobservation might be indicative of its better ability to escapedetection by the immune system. Nevertheless, serum from more animalssubjected to repeated administration of the DNA complex need to betested for the presence of neutralizing antibodies against the complexin order to conclude that immunogenicity is responsible for the failureof repeated injections in further lowering the cholesterol levels in theWatanabe rabbits.

EXAMPLE 5 Direct Injection of Complexed vs. Naked DNA into MuscleMethods

Three rats per experimental set were used in the experiments involvingdirect tissue injection of the DNA complex. One hundred micrograms ofnaked DNA containing the SV40-luciferase gene was injected into theliver and abdominal muscle of one of the animals. The same amount of theSV40-luciferase plasmid was complexed to poly-L-lysine and condensed asdescribed above and injected as well into the liver and abdominal muscleof the other two animals. The rats were sacrificed 48 hourspost-injection. A piece of liver and abdominal muscle were obtained forthe measurement of luciferase activity.

Results

Evaluation Of Direct Injections Of The DNA Complex Into The Liver AndMuscle Of Rats: The successful transfer of naked DNA into muscle cellsof mice by direct injection has been reported. Prolonged and high levelsof expression of a chimeric gene containing the Rous sarcoma virus (RSV)regulatory region linked to the luciferase cDNA were observed in theexperiments. We have investigated the advantages of using DNA complexedto poly-L-lysine and condensed over using free DNA, when DNA has to betransferred into the liver or muscle by direct injection. Three ratshave been used for these experiments. One hundred micrograms of nakedDNA encoding SV40-luciferase were injected into the liver and abdominalmuscle of one of the animals. The same amount of the pSV40-luciferaseplasmid complexed to poly-L-lysine and condensed as described above wasinjected as well into the liver and abdominal muscle of the other twoanimals. Rats were sacrificed 48 hours post-injection. A piece of liverand abdominal muscle were homogenized in lysis buffer and cell lysateswere analyzed for luciferase activity. All luciferase measurements wereperformed in triplicate, expressed as an average of the values andstandardized for total protein. FIG. 9 shows the integrated luciferaseunits per mg of protein in the two different sets of animals. Theefficiency of transfection of DNA complexed to poly-L-lysine andcondensed seems to be slightly higher when injected into the liver.However, it appears to result in a much higher efficiency whenintroduced into muscle tissue. We observe a 20-fold higher luciferaseactivity in the sample of muscle injected with the condensed DNAcompared to the one injected with naked DNA. We think that highlycondensed and packaged DNA may be protected against nucleases and may bemore stable. In addition, poly-L-lysines may increase the efficiency ofnuclear transport once inside the cell. First, the small size of thecomplex may allow its passage through nuclear pores and second, stringsof positively charged aminoacids as lysine and arginine are known to benuclear localization signals (NLS) in various nuclear proteins.Regarding the differences found between the response in the liver and inthe muscle, it is most probable that the characteristic interconnectedstructure of skeletal muscle cells makes them a better target for thepassive diffusion of DNA from cell to cell. This would easily allow thedistribution of the DNA complex along the muscle tissue and itstransport to the nuclei.

EXAMPLE 6 Direct Injection of Naked vs. Condensed DNA into the Brain:Gene Transfer of Retinal Ganglion Cells in Vivo Introduction

Insertion of foreign DNA into adult neurons has potentials for the studyof normal neuronal physiology and for the treatment of neural diseases.Gene transfer in neurons has been achieved using viral vectors, howeverit requires sophisticated methodologies and usually cells transfectedcan not be restricted to any particular type of neuron.

Axonal Retrograde transport is a continuous physiological process thathas been found to transport a large variety of different types ofmolecules. Many molecules are known to be incorporated into the axonlumen through endocytosis, whether they are adsorbed or fluid-phaseparticles. in the situation where axons have been severed, it ispostulated that soluble particles from the extracellular space candiffuse into the axon and move towards the soma.

In the present experiments we tested whether plasmid DNA naked orcondensed into a compact spheroid, applied to the cut end of retinalganglion cell axons in the optic nerve or to the tectum of the brain istransported back to the soma and expressed into protein.

Methods

Three plasmids under the control of one of three promoters which areeffective in a wide variety of eukariotic cell types were used:RSV-lacZ, CMV-lacZ and SV40-luc. They were prepared at differentconcentrations ranging from 1 to 20 μg/μl. pCMV-lacZ and pSV40-luc werecomplexed with poly-L-lysine (1:1) by Jose Carlos Perales (PNAS, 1994).

Assessment of retrograde transport of the plasmid complex to the retinalganglion cell somas was done using epifluorescence microscopyFITC-poly-L-lysine was used to form complexes with pCMV-lacz. To assessthe retrograde transport of pure plasmid, pRSV-lacZ was digested in onesite using Hind III. Biotin-dUTP was then linked to the 3'-OH ends ofpRSV-lacZ by reaction with Terminal dexynucleotidyl Transferase. Plasmidwas then precipitated and washed from free biotin-dtyrp and resuspendedat 2 μg/μl.

Adult Wistar rats were anesthetized and their optic nerves were exposed.1.5 μl of the plasmid solution (different concentrations and plasmids)was applied covering the Optic Nerve. Optic nerve axons were then cutavoiding the retinal blood supply. Another 1.5 μl of the same plasmidsolution was applied in soaked gelfoam. The conjunctiva was then closed.Same procedure was done in the contralateral eye using unspecificplasmid. Animals were sacrificed 3 days later. For direct injection intothe tectal area, nimals were anesthetized and injected stereoscopicallyinto the tectal area of the brain with naked DNA or condensed DNA.

For liquid β-galactosidase assays, retinas were kept at -70° C. untilthey were cell-lysed by repeated thawing and freezing. Tissue wascentrifuged at 12000 rpm for 2 min aiid the supernatant collected andanalyzed for protein content. Volumes containing 360 μg of protein wereincubated overnight at 37° C. in buffer A containing 15 mg/mlchlorophenol red B-D-galactopyranoside (CPRG). The absorbance wasrecorded.

For luciferase assays were done in lysis supernatants of retinas addedwith luciferase assay buffer. Samples were put into a luminometer whichwas injected with D-luciferin and then registered luminiscence.

For in situ β-galactasidase assays (for pRSV-lacZ and pCMV-lacZ) retinaswere fixed in 2% formaldehyde, 0.5%; glutaraldehyde, PBS for 30 min.,washed in PBS and incubated for 6 hrs at 37° C. in 1 mg/ml X-Gal, 4 mMpotassium ferrocyanide, 4 mM potassium ferricyanide, 2 mM MgCl₂, PBS pH7.3, 0.02% Nonidet p-40, 0.01% Deoxycholate. Tissue was then rinsed aridanalyzed immediately. Counts of blue labeled cells were made to estimatethe percentage of transfected cells.

Results

1) Administration of plasmid DNA to the cut end of rat optic axonsresults in its retrograde transport to the cell body. Double labeledfield (confocal microscopy) from a retina 2 days after administration ofFITC-poly-lysine/pCMV-lacZ complex at the cut end of the optic nerve andthen incubated in propidium iodide showed that FITC (green), Propidiumiodide (red) and the mixture of both nuclei double labeled (yellow),counted in randomized fields represented about 45% of the population ofretinal ganglion cells.

Microscopic fields taken at different magnifications showed blue coloredcells in the retinal ganglion cell layer following in situβ-galactosidase assay in retina. 20 μg/μl of pRSV-lacZ were administeredat cut optic nerve and comparison was made with contralateral eyetreated with pSV40-luc. Cells positive for β-galactosidase were noted tobe in the range size known only for ganglion cells in the retina. Thesecells were counted in randomized fields and were estimated to represent35% of total ganglion cells.

2) Plasmid DNA in retinal ganglion cells is expressed in a dosedependent manner and the condensed DNA is expressed at higherefficiency. Luciferase activity in retinas from rats whose severed opticnerves were administered with pSV40-luc at increasing concentrations, ascompared with retinas just axotomized, or treated with the non-specificplasmid pCMV-lacZ (1 μg/μl) showed concentration dependent increase inactivity of pSV40-luc.

The results of β-galactosidase activity in retinas from rats whosesevered optic nerves were administered with pCMV-lacZ, as compared withretinas just axotomized, or treated with non-specific plasmid pSV40-luc(10 μg/μl) showed that the highest activity was registered from themaximutn concentration of pCMV-lacZ. pCMV-lacZ complexed withpoly-lysine produced higher activity in β-galactosidase thannon-specific plasmid.

3) This method can be used in the transfer of specific genes to preciseneuronal types through their projections.

4) Intratectal injections of naked and polylysine condensed plasmid DNAcan achieve high levels of expression in the cell body of the neuronover 20 days. When the DNA is not condensed with poly-L-lysine the levelof expression returns to background after 10 days post-injection (FIG.10).

                  TABLE 101                                                       ______________________________________                                                                      Present                                                Wu et al.                                                                              Wagner et al. Invention*                                      ______________________________________                                        [DNA] mg/ml                                                                             ˜1  ˜0.01   ˜1                                    PO.sub.4 /NH.sub.3 Ratio                                                               ˜100 ˜1      ˜1.5                                  Buffer   150 mM NaCl                                                                              10 mM Hepes (pH 7),                                                                         Variable                                                        150 mM NaCl   [NaCl]                                      Compaction                                                                             Annealing  Direct Mixing Nucleation                                  Method                                                                        Structure Of                                                                           (Psi)      (Psi) or Unimolecular                                                                       Unimolecular                                The DNA                                                                       Complex                                                                       Size Of The                                                                            ≈200 nm                                                                          80 nm         ˜10 nm                                Complex                                                                       Diagnostic                                                                             Gel Retardation                                                                          Electron Microscopy                                                                         Circular                                    Tools                             Dichroism And                                                                 Electron                                                                      Microscopy                                  Expression in                                                                          Yes        No            Yes                                         vivo                                                                          Length Of                                                                              6 Days     --            At Least                                    Expression                        140 Days                                    ______________________________________                                         *Preferred Embodiment.                                                   

                  TABLE 102                                                       ______________________________________                                        Level Of Expression Of The PEPCK-hFIX Gene In The Livers Of Rats              Injected With The DNA Complex                                                 Rat #    Days After Injection                                                                       Units Of hFIX Activity                                  ______________________________________                                        1         2           0.040                                                   2         2           0.045                                                   3         4           0.045                                                   4         4           0.025                                                   5         6           0.330                                                   6         8           0.135                                                   7        12           0.160                                                   8        12           0.075                                                   9        32           0.125                                                   10       48           0.350                                                   11       72           0.005                                                   12       136          0.105                                                   ______________________________________                                    

                                      TABLE 103                                   __________________________________________________________________________    State Of DNA Or                                                                            Naked Eye (Or                                                    DNA/Polycation Complex                                                                     Turdimetry At 400 nm)                                                                     Circular Dichroism                                                                         Electron Microscopy                                                                        Absorbance At 260          __________________________________________________________________________                                                       nm                         Normal DNA (not                                                                            No turbidity. Clear                                                                       Normal DNA spectrum,                                                                       Very thin (about 1                                                                         This absorbance is                                                            the                        complexed).  solution.   i.e., maxima at 220 and                                                                    thick or less) and                                                                         reference for the                                                             other                                               269 nm; a minimum at                                                                       (about 300-1,000 nm)                                                                       states.                                             245 nm, a and a zero-point                                                                 fibers. (FIG. 1B).                                               crossover at 258 nm.                                 Condensed complex                                                                          Low turbidity. Almost                                                                     Identical to the spectrum                                                                  Individually isolated                                                                      About 20-30% of                                                               reference                  (caused by polycation).                                                                    clear solution.                                                                           of unbound (no poly-L-                                                                     spherical or toroidal                                                                      absorbance.                                         lysine) double stranded                                                                    structures. For DNA of                                           DNA in solution; positive                                                                  about 5 kb, the toroids are                                      maxima at 269 nm and                                                                       about 10-20 nm in external                                       very little contribution                                                                   diameter. Larger DNA, will                                       from the amide bond of                                                                     of course compact to form                                        the poly-L-lysine peptide                                                                  larger toroids. Electron                                         to the spectrum at 220 nm.                                                                 dense particles. No fibers.                                      (FIG. 1A).   (FIG. 1D).                              Relaxed complex (caused                                                                    No turbidity. Clear                                                                       Very difficult to                                                                          Rod-like fibers (usually                                                                   About 80-100% of           by excess salt).                                                                           solution.   differentiate from the                                                                     20 times the diameter of                                                                   reference absorbance.                               condensed form. The only                                                                   naked DNA fiber, i.e.,                                           difference is that there is                                                                usually 10-20 nm thick                                           some contribution from the                                                                 and longer than 60 nm) of                                        amide bond of the poly-L-                                                                  DNA and branched                                                 lysine peptide to the                                                                      toroidal structures of                                           spectrum at 220 nm.                                                                        increased size. (FIG. 1F).                                       (FIG. 1A).                                           Precipitated complex                                                                       DNA fibers in solution.                                                                   Flat spectrum. (FIG. 1I).                                                                  Complex of macroscopic                                                                     About 1% of reference      (caused by polycation if              (micrometer range)                                                                         absorbance.                insufficient salt).                   fibers.                                 Unimolecular aggregated                                                                    Highly variable from fine                                                                 Characteristic red-shift and                                                               Unimolecular toroidal                                                                      About 10-20% of                                                               reference                  complex.     particulate to highly turbid.                                                             positive ellipticity in the                                                                structures clumping                                                                        absorbance.                                         300-320 nm band.                                                                           together to fonn random                                          networks of heterogeneous                                                     size and shape.                                      Multimolec-ular                                                                            Clear.      Characteristic inversion in                                                                Isolated, multimolecular                                                                   About 100% of                                                                 reference                  aggregated complex       the spectrum maxima at                                                                     Toroidal structures                                                                        absorbance.                (caused by polycation if 269 nm to the negative.                                                                    variable size depending on              insufficient salt)..sup.1                                                                              Clear contribution from                                                                    the number of DNA                                                the amide bond of the                                                                      molecules condensed                                              poly-L-lysine peptide to                                                                   together. The size is                                            the spectrum at 220 nm.                                                                    usually approximately 10                                         (FIG. 1H).   to 70 times that of the                                                       unimolecular toroids. (See                                                    Wagner et al. and Shapiro                                                     et al). (FIG. 1G).                      __________________________________________________________________________     .sup.1 The DNA will aggregate into multimolecular complexes when the          concentration of polyL-lysine is increased suddenly in the DNA solution       (i.e., by adding polyL-lysine very rapidly to the vortexing solution of       DNA) or the direct mixing of DNA and polyL-lysine as in the method of         Shapiro also used by Wagner et al., Aggregation into multimolecular           complexes will be also the result of annealing both components                polyL-lysine and DNA)  #in a gradient of decreasing NaCl concentration        (i.e., the method of Wu and Wu).                                         

                                      TABLE 104                                   __________________________________________________________________________       DNA     Initial                                                                           Final                                                                             [DNA]                                                      Lys #                                                                            (% super-coiled)                                                                      [NaCl]                                                                            [NaCl]                                                                            (mg/ml)                                                                           Physical State**                                                                      Activity†                               __________________________________________________________________________     15*                                                                             CMV-βGal (50)                                                                    151.6                                                                             200 0.2 CD: ND  +                                                                     EL: ND                                                                        Turibidity: None                                        20*                                                                             MT-hGH (100)                                                                          0   267 0.85                                                                              CD: ND  -                                                                     EL: Relaxed                                                                   Turbidity: None                                         27*                                                                             PEPCK-hLDLR                                                                           178 439 1   CD: ND  +++                                               (100)               EL: Condensed                                                                 Turbidity: Low                                          56                                                                              RS-Tr (50)                                                                            803 1000                                                                              0.24                                                                              CD: ND  ND                                                                    EL: ND                                                                        Turbidity: None                                         56                                                                              CMV-βGal (50)                                                                    250 746 0.2 CD: ND  ND                                                                    EL: ND                                                                        Turbidity: Low                                          56*                                                                             PEPCK-hFIX                                                                            800 933 0.35                                                                              CD: ND  +++                                               (50)                EL: Condensed                                                                 Turbidity: Low                                          56*                                                                             PEPCK-hFIX                                                                            636 970 0.6 CD: ND  +++                                               (50)                EL: ND                                                                        Turbidity: Low                                         109*                                                                             CMV-βGal (50)                                                                    500 909 0.2 CD: +   +++                                                                   EL: ND                                                                        Turbidity: Low                                         109*                                                                             CMV-βGal (50)                                                                    689 1000                                                                              0.39                                                                              CD: ND  ND                                                                    EL: ND                                                                        Turbidity: None                                        109*                                                                             CMV-βGal (50)                                                                    616 1036                                                                              0.95                                                                              CD: ND  +++                                                                   EL: ND                                                                        Turbidity: Low                                         109*                                                                             CMV-βGal (50)                                                                    735 941 0.39                                                                              CD: ND  +++                                                                   EL: ND                                                                        Turbidity: Low                                         109*                                                                             CMV-βGal (50)                                                                    500 1031                                                                              0.7 CD: +   ND                                                                    EL: ND                                                                        Turbidity: Low                                         109                                                                              PEPCK-βGal (50)                                                                  617 1004                                                                              0.3 CD: ND  -                                                                     EL: ND                                                                        Turbidity: None                                        109*                                                                             PEPCK-βGal (50)                                                                  1085                                                                              1174                                                                              0.88                                                                              CD: ND  +++                                                                   EL: ND                                                                        Turbidity: Low                                         109*                                                                             PEPCK-hFIX                                                                            630 1063                                                                              08  CD: +   +++                                               (50)                EL: Condensed                                                                 Turbidity: Low                                         109                                                                              PEPCK-hFIX                                                                            636 970 0.26                                                                              CD: ND  ND                                                (50)                EL: Relaxed                                                                   Turbidity: None                                        109                                                                              PEPCK-hFIX                                                                            750 1120                                                                              0.8 CD: ND  ++                                                (50)                EL: Relaxed                                                                   Turbidity: Low                                         109*                                                                             PEPCK-hFIX                                                                            812 1098                                                                              0.7 CD: ND  +++                                               (50)                EL: Condensed                                                                 Turbidity: Low                                         109                                                                              PEPCK-hFIX                                                                            812 1127                                                                              0.69                                                                              CD: ND  ++                                                (50)                EL: Relaxed                                                                   Turbidity: None                                        109*                                                                             SV40-luc (80)                                                                         1091                                                                              1144                                                                              0.9 CD: ND  +++                                                                   EL: Condensed                                                                 Turbidity: Low                                         109*                                                                             SV40-luc (80)                                                                         1091                                                                              1144                                                                              0.9 CD: ND  +++                                                                   EL: Condensed                                                                 Turbidity: Low                                         109*                                                                             SV40-luc (80)                                                                         961 1140                                                                              0.88                                                                              CD: ND  +++                                                                   EL: ND                                                                        Turbidity: Low                                         109*                                                                             SV40-luc (80)                                                                         1091                                                                              1144                                                                              0.8 CD: ND  +++                                                                   EL: ND                                                                        Turbidity: Low                                         109                                                                              SV40-luc (80)                                                                         666 1000                                                                              0.19                                                                              CD: +   ND                                                                    EL: Relaxed                                                                   Turbidity: None                                        109*                                                                             SV40-luc (80)                                                                         961 1121                                                                              0.8 CD: ND  +++                                                                   EL: ND                                                                        Turbidity: None                                        109*                                                                             SV40-luc (80)                                                                         735 972 0.55                                                                              CD: ND  +++                                                                   EL: ND                                                                        Turbidity: Low                                         109*                                                                             Salmon sperm                                                                          900 1231                                                                              1   CD: ND  ND                                                DNA (0)             EL: ND                                                                        Turbidity: None                                        109                                                                              PEPCK-OTC (50)                                                                        774 948 0.9 CD: ND  ND                                                                    EL: ND                                                                        Turbidity: Low                                         123                                                                              SV40-luc (100)                                                                        719 1044                                                                              0.95                                                                              CD: ND  -                                                                     EL: Relaxed                                                                   Turbidity: None                                        123                                                                              SV40-luc (100)                                                                        905 1086                                                                              1   CD: ND  -                                                                     EL: Relaxed                                                                   Turbidity: None                                        123                                                                              SV40-luc (100)                                                                        689 1019                                                                              0.95                                                                              CD: ND  -                                                                     EL: ND                                                                        Turbidity: None                                        123                                                                              SV40-luc (100)                                                                        783 978 0.5 CD: ND  -                                                                     EL: ND                                                                        Turbidity: None                                        123                                                                              SV40-luc (100)                                                                        905 1149                                                                              0.57                                                                              CD: ND  -                                                                     EL: Relaxed                                                                   Turbidity: None                                        123*                                                                             CMV-βGal (ND)                                                                    825 1020                                                                              0.75                                                                              CD: ND  ND                                                                    EL: ND                                                                        Turbidity: None                                        150*                                                                             CMV-βGal (ND)                                                                    886 1077                                                                              0.5 CD: ND  +++                                                                   EL: Condensed                                                                 Turbidity: Low                                         150*                                                                             SV40-luc (80)                                                                         800 972 0.36                                                                              CD: ND  +++                                                                   EL: ND                                                                        Turbidity: Low                                         150                                                                              SV40-luc (80)                                                                         821 868 0.3 CD: Psi DNA                                                                           -                                                                     EL: Aggregated                                                                Turbidity: High                                        150*                                                                             SV40-luc (80)                                                                         821 968 0.3 CD: +   +++                                                                   EL: Condensed                                                                 Turbidity: Low                                         150                                                                              SV40-luc (80)                                                                         821 1071                                                                              0.3 CD: +   -                                                                     EL: Relaxed                                                                   Turbidity: None                                        240*                                                                             SV40-luc (80)                                                                         711 1125                                                                              1   CD: ND  +++                                                                   EL: Condensed                                                                 Turbidity: Low                                         240                                                                              SV40-luc (80)                                                                         711 1162                                                                              1   CD: ND  +                                                                     EL: Relaxed                                                                   Turbidity: Low                                         240                                                                              SV40-luc (80)                                                                         711 1280                                                                              1   CD: ND  -                                                                     EL: Relaxed                                                                   Turbidity: None                                        240                                                                              SV40-luc (80)                                                                         800 1007                                                                              1   CD: ND  -                                                                     EL: Aggregated                                                                Turbidity: High                                        240                                                                              T7-T7 (90)                                                                            708 1187                                                                              0.9 CD: +   ND                                                                    EL: Condensed                                                                 Turbidity: Low                                         240                                                                              T7-T7 (90)                                                                            708 1250                                                                              0.9 CD: +   -                                                                     EL: Relaxed                                                                   Turbidity: None                                        240                                                                              PEPCK-hLDLR                                                                           642 947 0.73                                                                              CD: Psi DNA                                                                           -                                                 (100)               EL: Aggregated                                                                Turbidity: None                                        240                                                                              PEPCK-OTC (50)                                                                        706 1174                                                                              0.35                                                                              CD: ND  ND                                                                    EL: ND                                                                        Turbidity: None                                        240                                                                              PEPCK-OTC (50)                                                                        898 1153                                                                              0.64                                                                              CD: ND  ND                                                                    EL: ND                                                                        Turbidity: None                                        __________________________________________________________________________     *Used in compiling Table 105.                                                 ND = Not determined.                                                          **Physical state of the DNA complex after polycation binding.                 1. When circular dichroism (CD) was determined the results are indicated      as follows: spectral changes due to the polycation condensation of DNA ar     insignificant (+); polycation condensation resulted in Psiform DNA due to     aggregation into multiomolecular complexes (either rodlike or toroidal)       (Psi DNA); appearance of an aberrant spectrum associated with a highly        aggregative state (-).                                                        2. Electron microscopic results have been indicated as follows: the           association of the polycation with the DNA results in aggregation into        complexes of increased size (>60 nm) (Aggregated); the structures             resulting from the condensation are rodlike relaxed toroids of increased      size (Relaxed); polycation binding results in proper condensatin (toroids     <30 nm in diameter) (Condensed). The number of properly condensed             structures (toroids) per microscopic field has not been determined.  #        There is approximately 3fold variation in the number of toroids visible i     the EL with different preparations of DNA complex.                            3. Turbidity measurements are based on visual inspection of the final         solution of DNA complex.                                                      †. A relative indication of the activity of the introduced gene        after introduction of the DNA complex:                                        hFIX (human factor IX) is measured by the western blot hybridization or b     a functional activity assay of rat plasma samples.                            βGal (galactosidase) activity is measured by in situ histochemistry      in fixed cells or tissue sections.                                            luc (luciferase) activity is measured using a specific enzyme activity        assay with tissue extracts.                                                   hLDLR (human LDL receptor) activity was measured indirectly after             determination of the total serum cholesterol levels in a rabbit model for     LDL receptor deficiency.                                                      hGH (human growth hormone) activity refers to a direct measurement of hGH     levels in the serum of animals transfected with the DNA complex. A            radioimmuno assay specific for hGH was used.                                  The activity is relative to all the experiment performed with the same        DNA. Not detectable activity after introduction of the DNA complex is         indicated by "--".                                                       

                                      TABLE 105                                   __________________________________________________________________________    Final [NaCl] = 555.75 + [DNA] mg/ml * 180.91 + log (lys length) *             __________________________________________________________________________    y18.32                                                                        Regression Statistics                                                         Multiple R:    0.881909585                                                    R Square:      0.777764515                                                    Adjusted R Square:                                                                           0.743574441                                                    Standard Error:                                                                             135.5087624                                                     Observations: 16                                                              Analysis of Variance                                                                 df     Sum of Squares                                                                        Mean Square                                                                            F     Significance F                           __________________________________________________________________________    Regression                                                                           2      835435.3166                                                                           417717.6583                                                                            22.748254                                                                           5.6792E-05                               Residual                                                                             13     238714.1209                                                                           18362.62469                                             Total  15     1074149.438                                                     __________________________________________________________________________           Coefficients                                                                         Standard Error                                                                        t Statistic                                                                            P-value                                                                             Lower 95%                                __________________________________________________________________________    Intercept                                                                            -555.757861                                                                          228.34416556                                                                          2.433887324                                                                             0.0279103                                                                          -1049.059922                             [DNA] mg/ml                                                                          180.9113279                                                                          125.4285365                                                                           1.442345841                                                                            0.1697596                                                                           -90.06049864                             log (lys length)                                                                     718.3211054                                                                          117.7844848                                                                           6.098605488                                                                            2.037E-05                                                                           463.8632453                              __________________________________________________________________________

                                      TABLE 106                                   __________________________________________________________________________    Estimated And Experimental Size Of Condensed DNA Complexes                                 Condensed Diameter (nm ± SD)                                               Electron                                                                            Hydrated Model (Partial                                                                  Hydrated Model (X-Ray                           DNA     Size (bp)                                                                          Microscope.sup.a                                                                    Specific Volume).sup.b                                                                   Diffraction Density).sup.c                      __________________________________________________________________________    PEPCK-hFIX                                                                            4,500                                                                              12.80 ± 1.56                                                                     18         22                                              PEPCK-hOTC                                                                            5,300                                                                              18.00 ± 1.83                                                                     20         23                                              SV40-luciferase                                                                       5,600                                                                              16.95 ± 3.50                                                                     20         24                                              PEPCK-CAT                                                                             5,800                                                                              16.30 ± 2.56                                                                     20         25                                              CMV-hLDLr                                                                             7,400                                                                              20.70 ± 2.60                                                                     22         26                                              φ29.sup.d                                                                         18,000                                                                             38.sup.e                                                                            40         47                                              __________________________________________________________________________     .sup.a measured diameter of at least 10 DNA complexes in a printed            photograph (× 240,000).                                                 .sup.b calculated diameter of a unimolecular DNA complex assuming a           condensed sphere. The partial specific volume of NaDNA was deemed to be       0.5 ml/g. The contribution of galactosylated polyL-lysine at a charge         ratio of 1:1 has been added. The molecular weight of DNA was calculated       based on an average molecular weight of 6,500 dalton/10 bp. The formula       used is:                                                                      DNA molecular weight (daltons)/6.023 × 10.sup.23 × 0.5 (ml/g)     = ml occupied by a molecule of DNA of X molecular weight. Diameter            obtained from the formula for the volume of a sphere.                         .sup.c calculated diameter of a unimolecular DNA complex assuming a           condensed sphere. The calculation assumed a hydrated density of 1.25 ±     0.1 g/ml as determined by Xray difraction. The contribution of a              galactosylated polyL-lysine at a charge ratio of 1:1 has been added. The      molecular weight of DNA was calculated based on an average molecular          weight of 6,500 dalton/10 bp. The formula is:                                 DNA molecular weight (daltons)/6.023 × 10.sup.23 × 1.25 (g/ml     = ml occupied by a molecule of DNA of X molecular weight. Diameter            obtained from the formula for the volume of a sphere.                         .sup.d from the literature.                                                   .sup.e the size to the phage prohead includes the protein outshell.      

We claim:
 1. A method for delivering an oligonucleotide to cells of amammal, comprising:a) providing:i) a target binding moiety consisting ofan antibody or a specific binding fragment thereof which binds to asecretory component of a polymeric immunoglobulin receptor present onthe surface of a liver or airway epithelium cell in a tissue of amammal; ii) a nucleic acid binding moiety consisting of a polycationicpolymer comprising positively charged amino acids; iii) an expressionvector comprising a promoter operably linked to an oligonucleotideencoding a chloride ion channel involved in cystic fibrosis; iv) arecipient mammal b) conjugating said target binding moiety to saidnucleic acid binding moiety to form a carrier; c) compacting saidcarrier with said expression vector in the presence of a chaotropic saltto a diameter of less than 30 nm to form a pharmaceutical composition;and d) administering said composition to said recipient mammal.
 2. Themethod of claim 1, wherein said cell is a mucosal epithelial cell. 3.The method of claim 1, wherein said promoter is a viral promoter.
 4. Themethod of claim 3, wherein said viral promoter is selected from thegroup consisting of the SV40 promoter, the MMTV promoter and the CMVpromoter.
 5. The method of claim 1 wherein said target binding moiety isan antibody which is capable of binding to the secretory component of amammalian polymeric immunoglobulin receptor.
 6. The method of claim 5wherein said antibody is a monoclonal antibody.
 7. The method of claim 1wherein said polycationic polymer comprising positively charged aminoacids is poly-L-lysine.
 8. The method of claim 1 wherein saidadministering comprises injection of said composition into saidrecipient animal.
 9. The method of claim 8 wherein said injection isintravenous.
 10. The method of claim 1 wherein said recipient animal isa human.
 11. The method of claim 1 further comprising followinginjection of said composition, examining said tissue containing saidpolymeric immunoglobulin receptor on their surface for the expression ofone or more of said gene products encoded by said expression vector. 12.A method for making a pharmaceutical composition suitable for deliveringan oligonucleotide to cells of a mammal, comprising:a) providing:i) anantibody or specific binding fragment thereof which binds to a secretorycomponent of a mammalian polymeric immunoglobulin receptor; ii) anucleic acid binding moiety consisting of a polycationic polymercomprising positively charged amino acids; and iii) an expression vectorcomprising a promoter operably linked to an oligonucleotide encoding oneor more gene products; b) conjugating said antibody or specific bindingfragment thereof to said nucleic acid binding moiety to form a carrier;and c) compacting said carrier with said expression vector in thepresence of a chaotropic salt to a diameter which is less than 30 nm toform a pharmaceutical composition.
 13. The method of claim 12, whereinsaid promoter is a viral promoter.
 14. The method of claim 13, whereinsaid viral promoter is selected from the group consisting of the SV40promoter, the MMTV promoter and the CMV promoter.
 15. The method ofclaim 12 wherein said antibody is a monoclonal antibody.
 16. The methodof claim 12 wherein said nucleic acid binding moiety is poly-L-lysine.17. The method of claim 1, whereby said one or more gene products areexpressed in said recipient mammal for at least 140 days.
 18. A methodfor delivering an oligonucleotide to cells of a mammal,comprising:administering a pharmaceutical composition to a recipientmammal, wherein said pharmaceutical composition comprises a compactedcomplex having a diameter of less than 30 nm of a carrier and anexpression vector, wherein the carrier comprises a target binding moietyconjugated to a nucleic acid binding moiety, wherein the target bindingmoiety consists of an antibody or specific binding fragment thereofcapable of binding to a secretory component of a mammalian polymericimmunoglobulin receptor, and the nucleic acid binding moiety consists ofa polycationic polymer comprising positively charged amino acids, andwherein the expression vector comprises a promoter operably linked to anoligonucleotide encoding a chloride ion channel involved in cysticfibrosis, whereby said one or more gene products are expressed by saidrecipient mammal.
 19. The method of claim 18, wherein said promoter is aviral promoter.
 20. The method of claim 19, wherein said viral promoteris selected from the group consisting of the SV40 promoter, the MMTVpromoter and the CMV promoter.
 21. The method of claim 18 wherein saidtarget binding moiety is an antibody directed against the secretorycomponent of a mammalian polymeric immunoglobulin receptor.
 22. Themethod of claim 21 wherein said antibody is a monoclonal antibody. 23.The method of claim 18 wherein said nucleic acid binding moiety ispoly-L-lysine.
 24. The method of claim 18 wherein said administeringcomprises injection of said composition into said recipient animal. 25.The method of claim 24 wherein said injection is intravenous.
 26. Themethod of claim 30 wherein said recipient animal is a human.
 27. Themethod of claim 24 further comprising following injection of saidcomposition, examining a tissue containing said polymeric immunoglobulinreceptor on its cells' surfaces for the expression of one or more ofsaid gene products encoded by said expression vector.
 28. The method ofclaim 27, wherein said expression of said gene products is for at least140 days.
 29. A compacted complex of a carrier and an expression vector,wherein the carrier comprises a target binding moiety conjugated to anucleic acid binding moiety, wherein the target binding moiety consistsof an antibody or specific binding fragment thereof which binds to asecretory component of a mammalian polymeric immunoglobulin receptor,and the nucleic acid binding moiety consists of a polycationic polymercomprising positively charged amino acids, wherein the expression vectorcomprises a promoter operably linked to an oligonucleotide encoding oneor more gene products, and wherein said complex is compacted to adiameter which is less than 30 nm.
 30. The compacted complex of claim29, wherein said promoter is a viral promoter.
 31. The compacted complexof claim 30, wherein said viral promoter is selected from the groupconsisting of the SV40 promoter, the MMTV promoter and the CMV promoter.32. The compacted complex of claim 29 wherein said target binding moietyis an antibody.
 33. The compacted complex of claim 32 wherein saidantibody is a monoclonal antibody.
 34. The compacted complex of claim 29wherein said polycationic polymer comprising positively charged aminoacids is poly-L-lysine.
 35. A compacted complex of a carrier and anexpression vector, made by the process of claim
 12. 36. The method ofclaim 12 wherein the oligonucleotide encodes a chloride ion channelinvolved in cystic fibrosis.
 37. The compacted complex of claim 29wherein the oligonucleotide encodes a chloride ion channel involved incystic fibrosis.